Application of plant ICAs gene and promoter thereof in cultivation of new varieties of stress-tolerant high-calcium crops

Abstract

The present invention relates to a recombinant construct comprising a first expression cassette comprising: a first nucleic acid molecule functional in a plant; the first nucleic acid molecule is selected from ICAs family genes; preferably, the first nucleic acid molecule codes an amino acid sequence as shown in any one of SEQ ID NO.1-9, or an amino acid sequence having at least 60% homology with the sequence as shown in any one of SEQ ID NO.1-9; and a first promoter heterologous operably linked to the first nucleic acid molecule. The application shows that by performing genetic manipulation on the ICAs gene of the plant and the promoter thereof, the Ca < 2 + > nutrient absorption of the plant can be promoted, so that the Ca < 2 + > content of the plant is increased, the tolerance of the plant to environmental stress is enhanced, and the method has important significance for cultivating new varieties of crops with high composite stress tolerance and new varieties of crops with high calcium nutrition.

Description

Technical Field

[0001] This invention relates to plant genetic engineering, and particularly to the application of plant ICAs genes and their promoters in the breeding of new stress-resistant, high-calcium crop varieties. Background Technology

[0002] Calcium ions (Ca 2+ As an important macronutrient element for plants, calcium plays a crucial role in plant growth, development, and defense mechanisms. 2+ It is absorbed through root cells, transported via the xylem, and distributed to the above-ground parts of the plant. Although plants can usually obtain calcium from relatively low external environments... 2+ It grows well at horizontal levels, but increases in Ca concentration within a moderate millimolecular range are beneficial. 2+ Concentrations can significantly enhance plant resistance to various abiotic and biotic stresses. However, similar to many other mineral nutrients, excessive calcium... 2+ It can cause toxicity and inhibit plant growth. Although Ca... 2+ It plays an important role in plant physiology, but root calcium 2+ The molecular mechanisms of absorption are still poorly understood.

[0003] Plants and animals Ca 2+ Comparative analysis of endorheic components revealed significant differences. Current evidence suggests that terrestrial plants possess relatively limited calcium. 2+ In contrast, animals possess a greater variety of pathways. During evolution, the genomes of Arabidopsis thaliana and other higher plants contain multiple groups of animal Ca... 2+ The homologs of these channels have been lost, including voltage-gated calcium channels (CaVs), transient receptor potential channels (TRPs), and ATP-gated purinergic receptor channels (P2XRs). Instead, homologs of cyclic nucleotide-gated channels (CNGCs), glutamate receptors (GLRs), and hyperosmolarly induced [CaVs] have been lost. 2+ ]( cyt OSCAs (Organizational System Components) – also known as Ca 2+ Permeability-gated cation channels (CSCs) and related gene families exhibit increased amplification in plants. Furthermore, nucleotide-binding and leucine-rich repeat receptors (NLRs) in plants can respond to pathogen effectors to form non-classical polyprotein Ca2+. 2+ The permeable channels, called resistances, operate on mechanisms distinctly different from those of animal NLRs. This difference suggests that plants may have evolved unique strategies to regulate Ca2+. 2+ Inflow. Additionally, evidence suggests that it promotes Ca2+ in plants. 2+ Other Ca inflow 2+The pathways remain to be discovered. For example, electrophysiological studies have revealed the presence of Ca in root cells. 2+ Permeability-free nonselective cation channels (CNCCs) that mediate Ca 2+ It is absorbed, but its molecular properties remain unclear.

[0004] Calcium ions (Ca 2+ Phosphorus is a macronutrient essential for plant growth and defense; however, the molecular mechanisms regulating its absorption from the soil remain largely unclear. Summary of the Invention

[0005] To address the technical problems existing in the prior art, the present invention proposes a recombinant construct comprising a first expression cassette, the first expression cassette comprising: a first nucleic acid molecule that is functional in plants; the first nucleic acid molecule being selected from ICAs family genes; and a first promoter that is heterologously operably linked to the first nucleic acid molecule.

[0006] The recombinant construct as described above, wherein the first nucleic acid molecule encodes an amino acid sequence as shown in any of SEQ ID NO. 1-9, or an amino acid sequence having at least 60% homology with any of the sequences shown in SEQ ID NO. 1-9.

[0007] The recombinant construct described above further includes a second expression cassette, wherein the second expression cassette includes: a second nucleic acid molecule that is functional in plants; the second nucleic acid molecule is selected from ICAs family genes; and a second promoter that is heterologously operably linked to the second nucleic acid molecule; wherein the second nucleic acid molecule is different from the first nucleic acid molecule.

[0008] In the recombinant construct described above, the second nucleic acid molecule encodes an amino acid sequence as shown in any of SEQ ID NO. 1-9, or an amino acid sequence having at least 60% homology with any of the sequences shown in SEQ ID NO. 1-9.

[0009] The recombinant construct described above, wherein the first promoter is the self-promoter of the ICAs gene in plants, or a plant recombinant promoter.

[0010] In the recombinant construct described above, the recombinant promoter is selected from one or more of the following group: CaMV35S promoter, Nos promoter, Ocs promoter, Actin promoter, and Ubiquitin promoter.

[0011] In the recombinant construct described above, the first promoter enables the expression of the first nucleic acid molecule in plant root cells.

[0012] The recombinant construct described above, wherein the second promoter is the plant's own promoter of the ICAs gene, or a plant recombinant promoter.

[0013] In the recombinant construct described above, the second promoter enables the expression of the second nucleic acid molecule in plant root cells.

[0014] In the recombinant construct described above, the first promoter may be the same as or different from the second promoter.

[0015] As described in any of the above recombinant constructs, the ICAs family genes include the eukaryotic cytochrome b561 domain and the extracellular dopamine β-monooxygenase N-terminal domain.

[0016] A carrier comprising any of the recombinant constructs described above.

[0017] The vectors described above, each of which includes one or more recombinant constructs.

[0018] A microorganism comprising any of the recombinant constructs described above, or comprising the vectors described above.

[0019] A plant, plant tissue, or plant cell that overexpresses one or more genes of the ICAs family relative to a wild-type plant, wherein the plant, plant tissue, or plant cell has resistance to adverse conditions.

[0020] A plant, plant tissue or plant cell comprising more of any of the recombinant constructs described above, or comprising the carriers described above, or comprising the microorganisms described above, relative to a wild-type plant, wherein the plant, plant tissue or plant cell has stress resistance properties.

[0021] Based on the plant, plant tissue or plant cell as described above, wherein the stress is an environment that causes biotic or abiotic stress to the plant.

[0022] Based on the plant, plant tissue or plant cell as described above, the biological stress environment is an environment in which plant pathogenic microorganisms are infected.

[0023] The microorganisms, based on the plant, plant tissue or plant cell as described above, are selected from one or more of the following groups: *Pseudomonas*, *Xanthomonas*, *Eurybacterium*, *Wildbacterium* and *Corynebacterium*.

[0024] Based on the plant, plant tissue or plant cell as described above, the abiotic stress environment is a high-salt environment, a high-temperature environment or a drought environment.

[0025] A food or feed composition comprising any of the plants, plant tissues or plant cells as described above.

[0026] The food or feed composition described above has a higher calcium content compared to wild-type plants, plant tissues, or plant cells.

[0027] This includes the use of ICAs family genes or their promoters, or recombinant constructs as described above, or vectors as described above, or microorganisms as described above in the cultivation of plants, plant tissues or plant cells that are resistant to abiotic stress or high calcium content.

[0028] In the application described above, the plant gene is overexpressed within the plant cells.

[0029] As described above, the plant is selected from one or more of the following plants: cereals, legumes, vegetables, tubers, fruits, fiber crops, oil crops, sugar crops, medicinal plants, forage crops, and ornamental plants;

[0030] As described above, the plant is selected from one or more of the following plants: Arabidopsis thaliana, Bryum simonii, rice, wheat, corn, barley, highland barley, oats, naked oats, sorghum, soybean, broad bean, pea, red bean, mung bean, radish, cabbage, celery, leek, garlic, onion, carrot, cucumber, lotus root, Jerusalem artichoke, sword bean, lettuce, cucumber, tomato, coriander, cilantro, potato, sweet potato, yam, pear, apple, plum, peach, plum, apricot, cherry, strawberry, crabapple, red date, citrus, cotton, flax, sisal, peanut, rapeseed, sunflower, sesame, sesame, camellia, sugarcane, beet, ginseng, osmanthus, honeysuckle, mint, mugwort, alfalfa, sweet clover, and milkvetch.

[0031] A method for preparing a plant, plant tissue, or plant cell that can resist abiotic stress or increased calcium content includes: overexpressing one or more genes or their promoters from the ICAs family in the crop; wherein the protein of the ICAs family gene includes a eukaryotic cytochrome b561 domain and an extracellular dopamine β-monooxygenase N-terminal domain.

[0032] The preparation method described above includes: transferring any of the recombinant constructs described above, or the vector described above, or the microorganism described above into a plant, a part of a plant tissue, or a plant cell; and regenerating a transgenic plant from the plant cell or part of a plant tissue, wherein, relative to the corresponding wild-type plant, the content of one or more ICAs family proteins in the transgenic plant, the part of the transgenic plant tissue, or the transgenic plant cell is increased.

[0033] A method for preparing a food or feed composition with high calcium content includes: overexpressing one or more genes or their promoters from the ICAs family in crops; wherein the proteins of the ICAs family genes include a eukaryotic cytochrome b561 domain and an extracellular dopamine β-monooxygenase N-terminal domain.

[0034] A method for preparing a plant, plant tissue, or plant cell sensitive to adverse environments includes: knocking down or reducing one or more genes or their promoters in the ICAs family of crops; wherein the proteins of the ICAs family genes include a eukaryotic cytochrome b561 domain and an extracellular dopamine β-monooxygenase N-terminal domain.

[0035] This application demonstrates that genetic manipulation of plant ICAs genes and their promoters can promote plant Ca2+. 2+ Nutrient absorption, thereby increasing plant calcium 2+ The content of calcium enhances the plant's tolerance to environmental stress, which is of great significance for breeding new varieties of crops that are highly resistant to complex stresses and new varieties of high-calcium nutrient crops. Attached Figure Description

[0036] The preferred embodiments of the present invention will now be described in further detail with reference to the accompanying drawings, wherein:

[0037] Figure 1 This is a homology analysis of plant ICA family proteins according to an embodiment of the present invention, including Arabidopsis thaliana (purple, AtICA1-9), rice (pink, OsICA1-10), alfalfa (dark blue, Medtr, MtICA1-7), sphagnum moss (yellow, XP, PpICA1-9), maize (red, ZmICA1-9), soybean (green, GLYMA, GmICA1-15), potato (sky blue, PGSC, StICA1-13), wheat (black, TraesCS, TaICA1-22), etc.;

[0038] Figure 2 a- Figure 2 n is the ion current induced by ICAs proteins in plants under a two-electrode voltage clamp (TEVC) hyperpolarization potential according to an embodiment of the present invention;

[0039] Figure 3 a- Figure 3 b is that oocytes expressing Arabidopsis AtICA1, AtICA2, AtICA3, or AtICA4 proteins according to an embodiment of the present invention exhibit significant Ca2+ expression under two-electrode voltage-clamp hyperpolarization potential. 2+ Inward current;

[0040] Figure 4 a- Figure 4 e represents the expression pattern of AtICAs genes in Arabidopsis thaliana and the subcellular localization of their encoded proteins according to an embodiment of the present invention;

[0041] Figure 5 a- Figure 5 g is an Arabidopsis thaliana ica1 / ica2 / ica3 / ica4 quadruple mutant that maintains Ca in the plant according to an embodiment of the present invention. 2+ Impaired homeostasis;

[0042] Figure 6 a- Figure 6 z is a quadruple mutant of ica according to an embodiment of the present invention that has defects in tolerance to biological and abiotic stresses;

[0043] Figure 7 a- Figure 7 g indicates that, according to one embodiment of the present invention, increased extracellular calcium ion concentration enhances the ability of wild-type Arabidopsis thaliana to cope with biological stress, while no enhancement was observed in the ica1 / 2 / 3 / 4 mutants;

[0044] Figure 8 a- Figure 8 e is an example of how the ICA gene, according to an embodiment of the present invention, affects plant gene expression profiles, depending on external Ca during salt stress. 2+ It plays a key role in horizontal transcriptional reprogramming. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] In the following detailed description, reference can be made to the accompanying drawings, which form part of this application and illustrate specific embodiments of the present application. In the drawings, similar reference numerals describe substantially similar components in different figures. Specific embodiments of the present application are described in sufficient detail below to enable those skilled in the art to implement the technical solutions of the present application. It should be understood that other embodiments or modifications to the embodiments of the present application may also be utilized.

[0047] The term "plant" as used in this article refers to a class of multicellular eukaryotic organisms in nature. Their cells have cell walls composed of cellulose, and the vast majority can produce nutrients through photosynthesis. They typically grow sessilely, absorbing water and minerals through their roots, and using photosynthetic pigments such as chlorophyll to convert light energy into chemical energy, transforming carbon dioxide and water into organic matter and oxygen to support their growth, development, and reproduction. Plants generally include major groups such as bryophytes, ferns, gymnosperms, and angiosperms. Based on their uses, plants can be broadly classified into edible plants, industrial plants, medicinal plants, ecological and agricultural plants, ornamental plants, and plants for special purposes.

[0048] The term "crops" as used in this article refers to all plants cultivated in agriculture, including food crops, cash crops, industrial raw material crops, forage crops, and medicinal crops. For example, rice, wheat, corn, and soybeans mentioned in this application are all food crops; corn is also used as a forage crop. Arabidopsis thaliana and sphagnum moss are often used as model plants in crop research.

[0049] The term "plant tissues" as used in this article refers to meristematic tissues, protective tissues, nutritive tissues, vascular tissues, and mechanical tissues. Specifically, plant tissues can include roots, stems, leaves, flowers, seeds, etc. In some embodiments, plant tissues can also be seeds, cells, or other tissues that have been cultured for a certain period of time.

[0050] The term "food" or "feed" as used herein refers to products prepared using plants, plant tissues, or plant cells. In this application, the product contains more calcium, providing more readily ingestible calcium to humans or animals.

[0051] The term "expression cassette" as used herein refers to a modified fusion gene containing a promoter and a specified gene. An expression cassette enables the specified gene to be expressed in designated cells under the drive of the promoter. In some embodiments, the specified gene is a plant ICAs family gene. In some embodiments, an expression cassette contains only one specified gene. In some embodiments, multiple different expression cassettes can be simultaneously transfected into the same plant. In other embodiments, an expression cassette includes multiple specified genes.

[0052] The "ICAs" mentioned in this article refer to IONIC CURRENT FAMILY A, a group of plant-specific proteins identified by the inventors through bioinformatics and electrophysiological screening. These proteins exhibit Ca2+ expression in heterologous expression systems. 2+Permeability-independent nonselective cation channel (CNCC) activity. ICAs are unique in the plant kingdom, and their partial amino acid sequences are shown in Table 1. Those skilled in the art should understand that there are many other plants that include ICA family genes; this application only lists a portion of them, and the listed portions are only for illustrating the technical solution of this application, and are not intended to limit the technical solution of this application.

[0053] This application designates AT4G12980 as AtICA1. Representing AtICA1, AlphaFold server predictions indicate that ICA proteins tend to form a dimer conformation, with each subunit containing five transmembrane helices and a putative pore domain. According to one embodiment of this application, ICA family genes include a eukaryotic cytochrome b561 domain and an extracellular dopamine β-monooxygenase N-terminal domain.

[0054] like Figure 1 As shown, ICA family genes are found in crops such as Arabidopsis thaliana, maize, rice, wheat, *Sphagnum moss*, soybean, and potato. ICAs are mainly expressed in root cells and located in the cytoplasmic membrane. The amino acid sequences shown in SEQ ID NO. 1-9 of this application are the amino acid sequences of ICA family genes in Arabidopsis thaliana. According to the inventors' comparison, the amino acid sequences of ICA genes in other plants have greater than 60% homology with the ICA sequences in Arabidopsis thaliana shown in SEQ ID NO. 1-9, and all include the eukaryotic cytochrome b561 domain and the N-terminal domain of extracellular dopamine β-monooxygenase. In some embodiments, the ICA family gene sequences in other plants have greater than 65%, or greater than 70%, or greater than 75%, or greater than 80%, or greater than 85%, or greater than 90% homology with at least one of the amino acid sequences shown in SEQ ID NO. 1-9. Here, homology refers to the degree of similarity in amino acid sequences, or it can be the percentage of identical amino acids in the total number of amino acids.

[0055] In Arabidopsis thaliana, external Ca 2+ Under limited or excessive concentrations, the ica1 / 2 / 3 / 4 quadruple mutants exhibited either high or reduced sensitivity, respectively. Furthermore, under normal Ca2+ conditions... 2+ Under these conditions, these mutants exhibit enhanced susceptibility to various abiotic and biotic stresses. The ica tetrap mutant lacks the previously identified Ca-mediated growth factor in the roots. 2+ The absorbed CNCC current resulted in lower calcium levels than wild-type (WT) plants and altered gene expression profiles.

[0056] The experimental results of this application indicate that plant ICAs may serve as components of CNCCs within plants, in non-optimal calcium environments. 2+ Under certain conditions, Ca is mediated2+ Absorption is crucial for root morphogenesis, and at the optimal Ca... 2+ Under these conditions, the plant developed a wide range of environmental stress tolerances. This study fills a significant gap in our understanding of nutrient absorption mechanisms and is of great importance for developing stress-resistant crops and calcium-rich food sources.

[0057] The term "overexpression" in this article refers to the expression of a specific gene at a higher protein level in modified plant cells compared to the wild-type plant. For example, in Arabidopsis thaliana, overexpressing AtICA1, AtICA2, AtICA3, and / or AtICA4 will result in higher levels of these proteins in the modified Arabidopsis compared to the wild-type. Overexpression of a specific gene typically enhances its function within the plant. For instance, overexpression of one or more ICAs family genes in a plant can enhance its resistance to abiotic and abiotic stresses.

[0058] In some embodiments, methods for overexpressing a specific gene in a plant include, but are not limited to: enhancing the function of the gene's promoter to increase its expression level; replacing the gene's promoter with a more powerful promoter; or introducing the gene's RNA or protein into plant cells.

[0059] In some embodiments, "adversity" as used herein refers to an environment that stresses the growth and development of a plant, which may include biotic or abiotic stresses on the plant. In some embodiments, abiotic stresses include, for example, drought or high salinity. In some embodiments, biotic stresses may be microorganisms such as bacteria, viruses, and fungi that cause disease in plants or affect their growth and development.

[0060] The term "two-electrode voltage clamp" as used in this article has the same meaning as "TEVC," and both "patch clamp" and "cell electrophysiological properties" are experimental methods for measuring these properties.

[0061] Two-electrode voltage clamps study the function of ion channels or receptors by controlling membrane potential. Two microelectrodes are inserted into the cell: one to measure the cell's membrane potential, and the other to inject current to compensate for changes in ion current caused by the opening of ion channels on the cell membrane, thus maintaining the membrane potential at a predetermined level. In this way, ion currents in cells at different membrane potentials can be precisely measured, allowing for the study of the function and properties of ion channels.

[0062] In some embodiments, the dual-electrode voltage clamp experimental method includes:

[0063] Preliminary experiment: Obtain the mRNA of a specific gene, reverse transcribe the mRNA using a specific reagent to obtain the cDNA sequence of the specific gene;

[0064] Microinjection: The cDNA is injected into Xenopus laevis oocytes using a microinjection system, and the oocytes are left to stand for 24-72 hours to allow the cDNA of the specific gene to be expressed in the oocytes.

[0065] Current-voltage recording: Oocytes expressing specific genes are placed in a two-electrode voltage clamp system, and current and voltage electrodes are inserted into the oocyte membrane. Ionic currents at different membrane sites are recorded in a perfusion solution containing specific ions, such as calcium, potassium, barium, and sodium ions.

[0066] Patch-clamp, also known as single-channel current recording, uses a specially designed glass micropipette to adhere to a cell surface patch and then voltage-clamps the patch, allowing measurement of the pA-level current generated by the opening of a single ion channel. It "clamps" a small section of the cell membrane at a fixed potential, and by recording the minute currents passing through the ion channels on the patch, it allows for the study of the molecular structure, functional properties, and roles of ion channels in cellular physiological and pathological processes. Patch-clamp experiments can be performed using methods known to those skilled in the art, and will not be elaborated upon in this application.

[0067] The "oocytes" mentioned in this article refer to oocytes from the African clawed frog (Xenopus laevis), which are an important expression system in molecular biology. By injecting DNA or mRNA into oocytes, the channel performance of proteins can be studied through electrophysiological experiments in a control manner.

[0068] The term "multiplex mutant" as used in this article has a similar meaning to "higher-order mutant," referring to a mutant that simultaneously exhibits mutations in multiple target genes or dysfunction in multiple target proteins. This includes several mutated target genes or several dysfunctional proteins, thus constituting a multiple mutant. For example, a mutant containing dysfunction in any three target proteins from AtICA1 to AtICA9 is a triple mutant; one containing dysfunction in any four target proteins from AtICA1 to AtICA9 is a quadruple mutant; one containing dysfunction in any five target proteins from AtICA1 to AtICA9 is a quintuple mutant; one containing dysfunction in any six target proteins from AtICA1 to AtICA9 is a hexaple mutant, and so on. The same principle applies to other plant species or other genes, and will not be elaborated upon here.

[0069] The "non-invasive micro-testing technology" described in this article is a precision analytical technique used for real-time, dynamic monitoring of the migration rate and direction of ions / molecules on the surface of living biological samples (such as cells, tissues, organs, or intact organisms). This non-invasive micro-testing technology can acquire ions (such as H+) between biological samples and their microenvironment under near-physiological conditions without damaging the sample structure, puncturing cells, or harming tissues. + K + Na + Ca 2+ It exchanges information with molecules such as Cl- or small molecules (such as O2, NO, H2O2, etc.), which can be used to study biological physiological functions, stress responses, etc.

[0070] The "bacterial flagellin" mentioned in this article refers to the granular proteins that make up bacterial flagellar fibers and are key pathogenic factors. Bacteria approach and infect the host through flagellin. Plants recognize specific fragments of flagellin through special receptors, triggering local and systemic defense responses.

[0071] This application relates to a recombinant construct comprising a first expression cassette, the first expression cassette comprising: a first nucleic acid molecule that is functional in a plant; the first nucleic acid molecule being selected from ICAs family genes; preferably, the first nucleic acid molecule encoding an amino acid sequence as shown in any of SEQ ID NO. 1-9, or an amino acid sequence having at least 60% homology with any of the sequences shown in SEQ ID NO. 1-9; and a first promoter heterologously operably linked to the first nucleic acid molecule.

[0072] In some embodiments, the recombinant construct further includes a second expression cassette, wherein the second expression cassette includes: a second nucleic acid molecule that is functional in plants; the second nucleic acid molecule is selected from ICAs family genes; preferably, the second nucleic acid molecule encodes an amino acid sequence as shown in any of SEQ ID NO. 1-9, or an amino acid sequence having at least 60% homology with any of the sequences shown in SEQ ID NO. 1-9; and a second promoter heterologously operably linked to the second nucleic acid molecule; wherein the second nucleic acid molecule is different from the first nucleic acid molecule.

[0073] In some embodiments, the first promoter and the second promoter are the plant's own promoters of the ICAs gene, or plant recombinant promoters; preferably, the recombinant promoter is selected from one or more of the following group: CaMV35S promoter, Nos promoter, Ocs promoter, Actin promoter, Ubiquitin promoter; preferably, the first promoter and the second promoter cause the first nucleic acid molecule and the second nucleic acid molecule to be expressed in plant root cells; preferably, the first promoter and the second promoter are the same or different.

[0074] In some embodiments, ICAs family genes include the eukaryotic cytochrome b561 domain and the N-terminal domain of extracellular dopamine β-monooxygenase.

[0075] This application further relates to a carrier comprising the above-described recombinant constructs; preferably, each of the carriers comprises one or more recombinant constructs.

[0076] This application further relates to a microorganism comprising the above-described recombinant construct or comprising the above-described vector.

[0077] This application further relates to a plant, plant tissue, or plant cell that, relative to a wild-type plant, contains more of the above-described recombinant constructs, or contains the above-described carriers, or contains the above-described microorganisms, and the plant, plant tissue, or plant cell has stress resistance properties.

[0078] In some embodiments, the stress is an environment that causes biotic or abiotic stress to the plant; preferably, the biotic stress environment is an environment infected by plant pathogenic microorganisms; more preferably, the microorganism is selected from one or more of the following groups: Pseudomonas, Xanthomonas, Erwinia, Wild Bacillus, and Corynebacterium; preferably, the abiotic stress environment is a high-salt environment, a high-temperature environment, or a drought environment.

[0079] Calcium ion accumulation occurs in the aforementioned plants, plant tissues, or plant cells, resulting in a higher calcium ion content in the cells or cells of specific tissues compared to the wild-type plant. Therefore, the modified plants, plant tissues, or plant cells described in this application can be used to prepare food or feed compositions containing more calcium.

[0080] This includes the application of ICAs family genes or their promoters, or the aforementioned recombinant constructs, or the aforementioned vectors, or the aforementioned microorganisms in cultivating plants, plant tissues, or plant cells that are resistant to abiotic stress or high calcium content. In some embodiments, the plant genes are overexpressed in the plant cells.

[0081] In some embodiments, the plant is selected from one or more of the following: cereals, legumes, vegetables, tubers, fruits, fiber crops, oil crops, sugar crops, medicinal plants, forage crops, and ornamental plants; preferably, the plant is selected from one or more of the following: Arabidopsis thaliana, Bryophytum comosum, rice, wheat, corn, barley, highland barley, oats, naked oats, sorghum, soybean, broad bean, pea, red bean, mung bean, radish, and cabbage. Celery, leeks, garlic, scallions, carrots, cucumbers, lotus root, Jerusalem artichokes, sword beans, lettuce, cucumbers, tomatoes, cilantro, coriander, potatoes, sweet potatoes, yams, pears, apples, plums, peaches, plums, apricots, cherries, strawberries, crabapples, red dates, citrus fruits, cotton, flax, sisal, peanuts, rapeseed, sunflowers, sesame, sesame seeds, camellias, sugarcane, beets, ginseng, osmanthus, honeysuckle, mint, mugwort, alfalfa, sweet clover, milkvetch.

[0082] This application relates to a method for preparing plants, plant tissues or plant cells that can resist abiotic stress or increased calcium content, comprising: overexpressing one or more genes or their promoters in the ICAs family of crops; wherein the proteins of the ICAs family genes include eukaryotic cytochrome b561 domains and extracellular dopamine β-monooxygenase N-terminal domains.

[0083] In some embodiments, the preparation method further includes: transferring the recombinant construct, vector, or microorganism described above into a plant, a part of a plant tissue, or a plant cell; and regenerating a transgenic plant from the plant cell or part of a plant tissue, wherein, relative to the corresponding wild-type plant, the transgenic plant, the part of a transgenic plant tissue, or the transgenic plant cell contains an increased amount of one or more ICAs family proteins.

[0084] This application relates to a method for preparing a food or feed composition with high calcium content, comprising: overexpressing one or more genes or their promoters from the ICAs family in crops; wherein the proteins of the ICAs family genes include a eukaryotic cytochrome b561 domain and an extracellular dopamine β-monooxygenase N-terminal domain.

[0085] This application relates to a method for preparing plants, plant tissues or plant cells that are sensitive to adverse environments, comprising: knocking down or reducing one or more genes or their promoters in the ICAs family of crops; wherein the proteins of the ICAs family genes include eukaryotic cytochrome b561 domains and extracellular dopamine β-monooxygenase N-terminal domains.

[0086] The technical solution of this application will be described below through specific embodiments. Those skilled in the art should understand that the following embodiments are only for clearer explanation of the technical solution of this application, and are not intended to limit the technical solution of this application.

[0087] Example 1: Identification of ICAs in the Plant Kingdom

[0088] In this embodiment, ICA genes from Arabidopsis thaliana were injected into oocytes of different Xenopus laevis species. AtCNGC18 was used as a positive control and water as a negative control. After standing for 24-36 hours, observation revealed that the ICA genes were localized to the cytoplasmic membrane. Under two-electrode voltage-clamp (TEVC) hyperpolarization potential, most ICA genes in almost all species induced strong inward currents, conventionally indicating either cation influx or anion efflux (e.g., ...). Figure 2 a, Figure 2 (as shown in b).

[0089] The applicant further used AtICA1-4 as an example to test the effect of ICAs family genes on barium ions (such as... Figure 2 c- Figure 2 (as shown in f), potassium ions (as shown in f) Figure 2 g- Figure 2 j、 Figure 2 o- Figure 2 (as shown in r), calcium ions (e.g.) Figure 2 k- Figure 2 (as shown in n) and H+ ions, hydroxide ions, etc. at different pH levels (e.g., n) Figure 2 The ion transport situation is shown in Figure s. Figure 2 c- Figure 3 As shown in Figure s, AtICA1-4 can transport the above ions. Therefore, it can be concluded that the ICAs family of genes are non-selective voltage-gated ion channels.

[0090] Analysis of ICAs family proteins revealed that they encode proteins with conserved eukaryotic cytochrome b561 domains and extracellular dopamine β-monooxygenase N-terminal (DOMON) domains, also known as Unknown Functional Domain 568 (DUF568), belonging to the CYBDOM protein superfamily. TEVC recordings of ICAs genes expressed in oocytes showed that almost all of them elicited significant ion currents, suggesting they may possess ion channel activity (e.g., ion channel activity). Figure 2 a, Figure 2 (as shown in b).

[0091] AtCYBDOMG (At5g54830) is an Arabidopsis CYBDOM protein containing a cytochrome b561 domain, two DOMON domains, and one DM13 domain; the Arabidopsis Cyt b561 protein (At5g38630 / AtCYB-1) contains only the cytochrome b561 domain; STROMAL CELL-DERIVED RECEPTOR 2 (SDR2) is the only human CYBDOM protein. These three proteins have structures similar to ICAs, but their expression in oocytes only induces background currents comparable to those in water-filled oocytes, without significant ion currents (e.g., ...). Figure 2 a, Figure 2 (as shown in b).

[0092] Given that the Arabidopsis Cyt b561 protein and human SDR2 function as oxidoreductases, transferring electrons from ascorbic acid to monodehydroascorbic acid or iron ion chelates, the electrophysiological results of this application indicate that the ionic currents recorded in oocytes are independent of these enzyme-mediated electron transfers. This also suggests that the ascorbic acid-dependent reductase activity of the Cyt b561 domain-containing protein does not induce activation of endogenous ion channels in oocytes.

[0093] ICAs are widely distributed in the plant kingdom. This application further analyzed the homology of ICAs from plants such as Arabidopsis thaliana, rice, alfalfa, *Sphagnum moss*, maize, soybean, and wheat, and found that ICA family genes are highly homologous among different species (e.g., Figure 1 (As shown).

[0094] This application further cloned OsICA3 (Os03g0194900) from rice, MtICA5 (Medtr8g009880) from alfalfa, and PpICA3 (XP024359865) from *Moss sphaerocephala*, and tested their ability to generate significant currents in oocytes (e.g., ...). Figure 2 a, Figure 2 (as shown in b).

[0095] Notably, all the proteins tested elicited strong ionic currents, indicating that ICAs are functionally conserved in mediating ion fluxes across different plant species.

[0096] Table 1 Protein information of ICAs in plants

[0097]

[0098]

[0099] Example 2: Electrophysiological characteristics of ICAs

[0100] To elucidate the electrophysiological characteristics of this potential family of ion channel components, this application uses Arabidopsis thaliana as an example, selecting AtICA1, AtICA2, AtICA3, and AtICA4 as representative members, and analyzes them using two-electrode voltage clamp (TEVC) recording.

[0101] First, this application determines whether ICAs mediate Ca2+ perfusion of oocytes using CaCl2 solution. 2+ Inflow. Compared to the weak background current produced by water-filled oocytes, oocytes expressing AtICA1, AtICA2, AtICA3, or AtICA4 exhibited significant inflow currents at hyperpolarization potentials, with the amplitudes ordered as follows: AtICA3 > AtICA1 > AtICA2 > AtICA4 (e.g., ...). Figure 3 a, Figure 3 (as shown in b).

[0102] Because the oocytes of the African clawed frog contain calcium 2+ Activated chloride channels (CaCCs), these channels in the cytoplasm Ca 2+ Concentration ([Ca 2+ i) Activated upon elevation, therefore these inward currents likely contain ICA-mediated Ca 2+ The influx is due to both inward and CaCC-mediated Cl- efflux. To separate the ICA-mediated components, this application adds a saturated concentration of the CaCC inhibitor 4,4'-diisothiocyanate stilbene-2,2'-disulfonic acid (DIDS) to the perfusion fluid. This significantly reduces the inward current (e.g., Figure 3 a, Figure 3 (as shown in b). However, small inward currents exhibiting nonrectification and weak voltage dependence still exist, and these currents can be generated by common Ca. 2+ Eliminated by the channel blocker LaCl3 (e.g.) Figure 3 a, Figure 3 (as shown in b). These findings indicate that ICAs promote Ca... 2+ internal flow.

[0103] In summary, the results of this embodiment demonstrate that ICAs mediate the flow of both divalent and monovalent cations, including Ca. 2+ Ba 2+ Mg 2 + K + and Na + These findings identify ICAs as potential components of non-selective cation channels (CNCCs).

[0104] Example 3: Expression Patterns and Protein Localization

[0105] To elucidate the physiological functions of ICA proteins, this application uses Arabidopsis thaliana as an example to examine the expression patterns of six AtICAs genes in Arabidopsis thaliana and the subcellular localization of their encoded proteins.

[0106] This application constructed transgenic Arabidopsis plants that expressed β-glucuronidase (GUS) reporter genes under the control of their respective AtICA1-AtICA6 promoters. For AtICA1 to AtICA5, GUS staining was mainly observed in the roots and floral organs, with AtICA1 and AtICA5 showing relatively weak expression. In contrast, AtICA6 showed widespread expression throughout the entire plant (e.g., ...). Figure 4 a, Figure 4 (as shown in b).

[0107] To more sensitively detect protein localization, this application created transgenic plants expressing AtICA1-YFP, AtICA2-YFP, AtICA3-YFP, and AtICA4-YFP fusion proteins under the control of their respective native promoters. The fluorescence signals of AtICA1-YFP and AtICA2-YFP were mainly detected in the root vascular tissue, while their expression was weaker in all cell layers outside the endodermis (e.g., ...). Figure 4 c. Figure 4 (as shown in d). Conversely, the fluorescence signals of AtICA3-YFP and AtICA4-YFP were distributed in all cell layers of the root (as shown in d). Figure 4 (c, d). Further analysis confirmed that AtICA1, AtICA2, AtICA3, and AtICA4 proteins are located on the cytoplasmic membrane of root protoplast cells (e.g., ...). Figure 4 (as shown in e).

[0108] Example 4: The effect of the ica mutant on external Ca 2+ response

[0109] To characterize the phenotypes associated with AtICA proteins, this application used CRISPR / Cas9 technology to knock out AtICAs members based on their expression patterns, generating higher-order ica mutants (such as...). Figure 5 a- Figure 5 (as shown in d). Figure 5 a- Figure 5 In section c, different knockout patterns of single-gene mutants of AtICA1-6 are shown. Figure 5 d shows the mutation patterns of each ICA gene in the multiple mutants. For example, the multiple mutants of ica1 / 2 / 3 / 4-1 include simultaneous mutations of ica1-1, ica2-1, ica3-1 and ica4-2. Here, ica1-1 refers to the ica1-1 mutation pattern as shown in 5a, and the mutation patterns of ica2-1, etc. are similar and will not be described again here.

[0110] Experimental results showed that the two independent ica1 / ica2 / ica3 / ica4 quadruple mutant series (named ica1 / 2 / 3 / 4-1 and ica1 / 2 / 3 / 4-2, respectively) exhibited similarities to Ca 2+ Related phenotypes.

[0111] Under normal growth conditions, these mutants did not exhibit obvious developmental abnormalities (e.g., Figure 5 (as shown in e). However, when exposed to different Ca... 2+ At certain concentrations, the mutants react differently than the wild-type (WT) plants (e.g. Figure 5 f). In low Ca 2+ Under these conditions (0.05 or 0.1 mM), compared with normal Ca 2+ Compared to the levels (1.0 or 5.0 mM), both the wild type and the ica quadruple mutant showed inhibition of primary root growth (e.g., Figure 5 f、 Figure 5 (as shown in g). However, the inhibition level of the quadruple mutant was significantly more pronounced, indicating that the ica quadruple mutant was more effective against low Ca2+. 2+ High sensitivity (e.g.) Figure 5 f、 Figure 5 (as shown in g). Conversely, in high Ca... 2+ Under these conditions (10.0 or 18.0 mM), taproot growth was inhibited in wild-type plants, but this effect was less pronounced in the quadruple mutant, suggesting that high Ca2+ levels may hinder root development. 2+ Reduced sensitivity (e.g.) Figure 5 f、 Figure 5 (as shown in g).

[0112] These genetic findings, combined with the preferential expression of the ICA gene in root cells and its role in Ca2+, indicate that... 2+ Its role in transport suggests that AtICA protein promotes Ca2+ transport. 2+ Absorbed, and may help maintain calcium in plants. 2+ Steady state.

[0113] Example 5: Reduced stress tolerance in ica mutants

[0114] Calcium ions (Ca 2+ As an important macronutrient, calcium is crucial for various stress responses in plants. Although the icas tetrap mutant exhibits normal calcium levels... 2+ No observable developmental defects were observed at the level of [specific level], and this application further evaluated their resilience.

[0115] When exposed to abiotic stress conditions, such as 85 mM NaCl, 250 mM mannitol, or heat treatment at 45°C, the ica tetrad mutant exhibited more severe inhibition in taproot and seedling growth compared to the wild-type (WT) plant. Figure 6 a- Figure 6 (as shown in d). These results indicate that the ica quadruple mutant has defects in tolerance to abiotic stresses.

[0116] This application further investigated the role of AtICAs genes in biotic stress resistance. Treatment with the bacterial flagellin epitope flg22 triggered a pathogen-associated molecular pattern-induced immune response (PTI), leading to inhibited growth in wild-type plants. However, this effect was mitigated in the ica quadruple mutant, manifested by a longer taproot, indicating reduced PTI intensity (e.g., Figure 6 h- Figure 6 Consistent with this finding, when infected by the bacterial pathogen *Pseudomonas tomatoides* DC3000 (PstDC3000), the ica tetrad mutant developed more severe chlorophyll degradation symptoms (e.g., as shown in i) compared to the wild-type plant. Figure 6 e- Figure 6 (as shown in f), and exhibited higher bacterial counts (e.g. Figure 6 (as shown in g). Similarly, inoculation with the fungal pathogen *Botrytis cinerea* resulted in the *ica* tetrad mutant producing larger lesions, indicating increased susceptibility to the pathogen (e.g., as shown in g). Figure 6 j- Figure 6 (as shown in k).

[0117] Consistent with the reduced stress tolerance observed in the ica quadruple mutant, the promoter activities of AtICA1, AtICA2, AtICA3, and AtICA4 were strongly induced by treatment with NaCl and flg22. Figure 5 k,l).

[0118] To determine whether AtICA1, AtICA2, AtICA3, and AtICA4 co-regulate stress tolerance, this application exposed triplet mutants (ica1 / 2 / 3, ica1 / 2 / 4, ica1 / 3 / 4, and ica2 / 3 / 4) to NaCl, mannitol, flg22, and Botrytis cinerea. These triplet mutants exhibited stress responses similar to those of wild-type plants, indicating redundancy in the functions of the AtICA1, AtICA2, AtICA3, and AtICA4 genes (e.g., ...). Figure 6 n- Figure 6(As shown in y). The ica1 / 2 / 3 / 4 / 5 / 6 hexaple mutant exhibits a high salt tolerance defect similar to that of the ica1 / 2 / 3 / 4 quadruple mutant, indicating that AtICA5 and AtICA6 contribute minimally to the stress tolerance mechanism (e.g., Figure 6 (as shown in z).

[0119] AtICA1, AtICA2, AtICA3, AtICA4, and AtICA5 and AtICA6 mutants exhibit broad-spectrum intolerance to both biotic and abiotic stresses. These results indicate that, under normal external conditions, Ca... 2+ Under certain conditions, AtICA1-AtICA6 play an active role in modulating responses to a wide range of environmental stresses.

[0120] Example 6: ICAs promote Ca 2+ absorb

[0121] Although patch-clamp recordings of root cells measured Ca2+ via the symplastic pathway 2+ Influx occurs, but in root regions lacking continuous Casparian strips between endothelial cells, the apoplast pathway also promotes Ca2+ flow. 2+ The movement from the soil to the xylem.

[0122] To measure the total Ca in the root elongation zone 2+ For internal flow, this application utilizes non-destructive microtesting (NMT) technology. Experimental results show that, without NaCl treatment, the average net Ca on the WT root surface... 2+ The influx is higher than that on the surface of the ica1 / 2 / 3 / 4 mutant (e.g. Figure 7 a- Figure 7 (as shown in b). NaCl treatment suppressed the average net Ca2+ in both genotypes. 2+ Influx, but maintained the differences between WT and ica1 / 2 / 3 / 4 mutants (e.g. Figure 7 c- Figure 7 (as shown in d).

[0123] Furthermore, this application determined the calcium content in WT and ica1 / 2 / 3 / 4 mutants, and found that under normal growth conditions, the calcium content of ica1 / 2 / 3 / 4 mutants was indeed lower than that of WT plants. NaCl treatment reduced the calcium content in WT and ica1 / 2 / 3 / 4 mutants, but differences between WT and ica1 / 2 / 3 / 4 mutants still existed (e.g., Figure 7 (as shown in e).

[0124] Therefore, non-invasive experiments demonstrated reduced calcium ion uptake in the ica1 / 2 / 3 / 4 mutants. These results collectively indicate that ICAs in root cells promote calcium absorption. 2+ absorb.

[0125] In suitable Ca2+ Within the range, add external Ca 2+ This usually enhances the plant's resistance to stress. If AtICAs mediate external Ca... 2+ Absorption, this effect should be weakened in the ica1 / 2 / 3 / 4 mutants. When external Ca 2+ When the NaCl level was increased from 1.5 mM to 5.0 mM, WT plants showed enhanced NaCl tolerance, evidenced by longer taproots (e.g., Figure 6 f- Figure 6 (as shown in g). However, this Ca 2+ The induced enhancement of NaCl tolerance was weakened in the ica1 / 2 / 3 / 4 mutants, which consistently exhibited reduced NaCl tolerance (e.g., Figure 6 f- Figure 6 (as shown in g).

[0126] Example 7: ICAs Affect Gene Expression Profiles

[0127] To detect whether gene expression profiles are altered in the ica quadruple mutant, this application investigates gene expression patterns in low-Ca2+ mutants. 2+ (0.1mM Ca 2 + ) or normal Ca 2+ (1.5mM Ca 2+ RNA sequencing (RNA-seq) analysis was performed on seedlings grown under certain conditions, with / without high salt (±NaCl) stress.

[0128] In low Ca2+ without salt stress 2+ Under certain conditions, compared to the wild type, 250 genes were upregulated and 112 genes were downregulated in the ica quadruple mutant (e.g., Figure 8 As shown in a). Gene Ontology (GO) enrichment analysis indicated that these differentially expressed genes (DEGs) were mainly associated with "photosynthesis", "electron transport chain", "phenylpropanin biosynthesis", and "suberin biosynthesis" (e.g., Figure 8 (as shown in e).

[0129] When exposed to low Ca 2+ Under high salt stress, compared to the wild type, the ica quadruple mutant showed 460 upregulated genes and 165 downregulated genes (e.g., ...). Figure 8 (as shown in b). Interestingly, GO enrichment analysis failed to identify any significantly enriched biological processes under these conditions, suggesting the possible presence of dysregulated stress responses.

[0130] In normal calcium levels without salt stress 2+ Under certain conditions, 282 genes were upregulated and 169 genes were downregulated in the ica quadruple mutant (e.g., Figure 8(As shown in c). GO analysis revealed top-level DEG clusters (such as...) associated with "drug catabolic processes," "negative regulation of catalytic activity," and "antibiotic catabolic processes." Figure 8 (as shown in e).

[0131] The most significant transcriptional changes occur in normal calcium. 2+ In conjunction with high salt stress, 808 genes were upregulated and 312 genes were downregulated in the ica quadruple mutant (e.g., Figure 8 (As shown in d). GO enrichment analysis highlighted significant clusters associated with "response to toxic substances", "detoxification", and "drug catabolism".

[0132] Although these stress response pathways are activated, the ica quadruple mutant exhibits normal external Ca 2+ Reduced salt tolerance under certain conditions indicates inefficient use of these detoxification mechanisms (e.g., Figure 8 (as shown in e).

[0133] These RNA-seq results indicate that the ICA gene depends on external calcium during salt stress. 2+ It plays a crucial role in horizontal transcriptional reprogramming. Increased external Ca 2+ Levels of calcium-dependent ICA gene expression are induced, and this expression is further regulated by salt stress. Under normal calcium conditions... 2+ The significant increase in DEGs under salt stress conditions highlights the role of the ICA gene in integrating external Ca2+. 2+ Importance of levels and salt stress response.

[0134] The above embodiments are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the scope of the invention. Therefore, all equivalent technical solutions should also fall within the scope of the invention.

Claims

1. A recombinant construct comprising a first expression box, the first expression box comprising: The first functional nucleic acid molecule in plants; The first nucleic acid molecule is selected from ICAs family genes; preferably, the first nucleic acid molecule encodes an amino acid sequence as shown in any one of SEQ ID NO. 1-9, or an amino acid sequence having at least 60% homology with any one of the sequences shown in SEQ ID NO. 1-9; and A first promoter that is heterologously operably linked to the first nucleic acid molecule.

2. The recombinant construct of claim 1, further comprising a second expression cassette, wherein, The second expression box includes: A functional second nucleic acid molecule in a plant; the second nucleic acid molecule is selected from ICAs family genes; preferably, the second nucleic acid molecule encodes an amino acid sequence as shown in any one of SEQ ID NO. 1-9, or an amino acid sequence having at least 60% homology with any one of the sequences shown in SEQ ID NO. 1-9; and A second promoter that is heterologously and operatively linked to the second nucleic acid molecule; The second nucleic acid molecule is different from the first nucleic acid molecule.

3. The recombinant construct according to claim 1, wherein the first promoter and the second promoter are the self-promoters of the ICAs gene in plants, or are plant recombinant promoters; Preferably, the recombinant promoter is selected from one or more of the following group: CaMV35S promoter, Nos promoter, Ocs promoter, Actin promoter, and Ubiquitin promoter; Preferably, the first promoter and the second promoter enable the expression of the first nucleic acid molecule and the second nucleic acid molecule in plant root cells; Preferably, the first promoter is the same as or different from the second promoter.

4. The recombinant construct according to any one of claims 1-3, wherein the ICAs family gene comprises a eukaryotic cytochrome b561 domain and an extracellular dopamine β-monooxygenase N-terminal domain.

5. A plant, plant tissue, or plant cell that overexpresses one or more genes of the ICAs family relative to a wild-type plant, wherein the plant, plant tissue, or plant cell has stress resistance properties.

6. The plant, plant tissue or plant cell according to claim 5, wherein the stress is an environment that causes biotic or abiotic stress to the plant; Preferably, the biological stress environment is a plant pathogen infection environment; More preferably, the microorganism is selected from one or more of the following groups: Pseudomonas, Xanthomonas, Erwinia, Wildbacterium, and Corynebacterium; Preferably, the abiotic stress environment is a high-salt environment, a high-temperature environment, or a drought environment.

7. A food or feed composition comprising the plant, plant tissue or plant cells as described in any one of claims 5-6.

8. The use of genes comprising the ICAs family or their promoters, or recombinant constructs as described in any one of claims 1-4, in the cultivation of plants, plant tissues or plant cells resistant to abiotic stress or high calcium content.

9. A method for preparing a plant, plant tissue, or plant cell that can resist abiotic stress or increased calcium content, comprising: Overexpression of one or more genes or their promoters from the ICAs family in crops; The proteins of the ICAs family genes include the eukaryotic cytochrome b561 domain and the N-terminal domain of extracellular dopamine β-monooxygenase.

10. The preparation method according to claim 9, comprising: Transform the recombinant construct as described in any one of claims 1-4 into a plant, a part of a plant tissue, or a plant cell; as well as Genetically modified plants are regenerated from plant cells or parts of plant tissues; Among them, compared with the corresponding wild-type plant, the content of one or more ICAs family proteins in the transgenic plant, parts of the transgenic plant tissues, or cells of the transgenic plant is increased.

11. A method for preparing a food or feed composition with a high calcium content, comprising: Overexpression of one or more genes in the ICAs family of crops with fiery promoters; The proteins of the ICAs family genes include the eukaryotic cytochrome b561 domain and the N-terminal domain of extracellular dopamine β-monooxygenase.

12. A method for preparing a plant, a portion of a plant tissue, or plant cells that are sensitive to adverse environmental conditions, comprising: Knock down or remove one or more genes or their promoters from the ICAs family of crops; The proteins of the ICAs family genes include the eukaryotic cytochrome b561 domain and the N-terminal domain of extracellular dopamine β-monooxygenase.

Images