Methods for modulating magnesium ion uptake and transport in plants

Abstract

The present invention relates to methods of modulating magnesium ion uptake and transport in plants. Specifically, a method of modulating magnesium ion uptake and transport in plants, ameliorating conditions caused by magnesium deficiency or excess in plants, or modulating plant growth, comprising modulating the expression or activity of aquaporins is provided. Aquaporins have been found for the first time to have a function in Mg 2+ uptake and transport.

Description

TECHNICAL FIELD

[0001] The present application relates to plant mineral absorption and transport, in particular to the use of aquaporins in regulating plant magnesium ion absorption and transport. BACKGROUND

[0002] Aquaporins (AQP) are membrane intrinsic proteins on cell membranes that can selectively and efficiently transport water molecules, belonging to the MIP (major intrinsic protein) superfamily. The AQP family has highly conserved structural features and functional domains. Usually in the form of tetramer, a few in the form of dimer or monomer. For tetrameric AQP, each monomer forms an independent water channel.

[0003] Plant aquaporins are divided into seven subgroups according to their sequences: plasmamembrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), Nodulin 26-like intrinsic proteins (NIPs) and small and basic intrinsic proteins (SIPs), GlpF-like intrinsic proteins (GIPs), HIPs (hybrid intrinsic proteins) and XIPs (Xintrinsic proteins, XIPs). Among them, PIP-type aquaporins are divided into PIP1, 2 and 3 subgroups.

[0004] AQP almost exists in all organs and tissues of plants, and is preferentially expressed in related cells and tissues involved in water transport, such as vascular tissue, vessels, xylem parenchyma cells and phloem. In addition to transporting water, plant AQP can also transport many other small molecules, such as neutral small molecules glycerol, H2O2, urea, NH3, CO2, silicic acid, boric acid and formamide, etc. AQP has multiple biological functions, participates in anther dehiscence, pollen recognition, pollen germination and flower opening, participates in seed maturation and germination, stomatal movement and CO2 transmembrane movement, etc.

[0005] The expression of plant AQP is regulated by abiotic stress, such as drought, cold, salt stress and waterlogging, as well as ABA and other signal molecules. The activity regulation mode of plant AQP involves phosphorylation, heterodimerization, glycosylation and methylation of AQP, as well as proton gradient (pH) and Ca 2+The function of aquaporins in Mg 2+ absorption and transport has not been reported. If the plant yield can be significantly increased by increasing the plant's absorption of minerals, its production potential can be stimulated. SUMMARY

[0006] Aquaporins mainly play a major role in the transmembrane transport of water. The present application finds that aquaporins not only have water transport function, but also have Mg2+ absorption and transport function. Therefore, the combination of water transport and mineral Mg transport is undoubtedly very beneficial to the growth and development of plants, which will greatly improve the growth efficiency, increase the biomass and yield.

[0007] The first aspect of the present application provides a method for regulating the absorption and transport of Mg2+ by plants, improving the condition of plants caused by Mg deficiency or high Mg, or regulating the growth of plants, comprising: regulating the expression or activity of aquaporins.

[0008] In one or more embodiments, the method up-regulates the expression or activity of aquaporins; thereby up-regulating the absorption and transport of Mg2+ by plants, improving the condition of plants caused by Mg deficiency, or promoting the growth of plants.

[0009] In one or more embodiments, the method down-regulates the expression or activity of aquaporins; thereby down-regulating the absorption and transport of Mg2+ by plants, improving the condition of plants caused by high Mg, or inhibiting the growth of plants.

[0010] In one or more embodiments, the plant is a Euphorbiales plant; preferably a Euphorbiaceae plant; more preferably a Manihot plant.

[0011] In one or more embodiments, the aquaporin is selected from one or more of the following: PIP, TIP, NIP, SIP, GIP, HIP and XIP.

[0012] In one or more embodiments, the aquaporin is selected from one or more of PIP1, PIP2 and PIP3.

[0013] In one or more embodiments, the aquaporin or its gene is derived from a Euphorbiales plant, preferably from a Euphorbiaceae plant, more preferably from a Manihot plant.

[0014] In one or more embodiments, the amino acid sequence of the aquaporin is selected from one or more of the following:

[0015] (a) a polypeptide having the sequence shown in SEQ ID NO: 1;

[0016] (b) a polypeptide derived from (a) by substitution, deletion, or addition of one or more (e.g., 1-20; preferably 1-10; more preferably 1-5) amino acid residues of the sequence set forth in SEQ ID NO: 1, and having a function of the polypeptide of (a); or

[0017] (c) a polypeptide derived from (a) having 90% (preferably 93%; more preferably 95% or 98%) or more homology to the polypeptide sequence of (a) and having a function of the polypeptide of (a).

[0018] In one or more embodiments, the nucleic acid sequence encoding the aquaporin is selected from one or more of the following:

[0019] (1) a polynucleotide encoding a polypeptide as set forth in SEQ ID NO: 1;

[0020] (2) a polynucleotide as set forth in SEQ ID NO: 2 or a polynucleotide having 80% (preferably 90%; more preferably 95% or 98%) or more homology thereto;

[0021] (3) a polynucleotide truncated at the 5' end and / or 3' end of the polynucleotide set forth in SEQ ID NO: 2 by 1-60 (preferably 1-30, more preferably 1-10) nucleotides;

[0022] (4) a polynucleotide complementary to any one of the polynucleotides of (1)-(3).

[0023] In one or more embodiments, the method of upregulating the expression or activity of an aquaporin in a plant comprises:

[0024] (1) introducing an aquaporin gene into a plant to obtain a transformed plant; and / or

[0025] (2) contacting an aquaporin gene or a facilitator of the protein with a plant.

[0026] In one or more embodiments, the facilitator comprises an agent capable of phosphorylating, hetero-oligomerizing, glycosylating, and methylating the aquaporin or an agent that increases the proton gradient (pH), Ca 2+ of the cellular environment.

[0027] In one or more embodiments, the aquaporin gene comprises a cDNA sequence, a genomic sequence, or a combination thereof.

[0028] In one or more embodiments, the expression of the aquaporin is driven by a 35S promoter.

[0029] In one or more embodiments, the method of upregulating the expression of an aquaporin in a plant comprises:

[0030] (1) providing Agrobacterium carrying a nucleic acid construct comprising an aquaporin gene,

[0031] (2) contacting a cell or tissue or organ of a plant with the Agrobacterium of step (1), thereby transferring the nucleic acid construct into the plant tissue or organ.

[0032] In one or more embodiments, the nucleic acid construct is an expression vector or an integration vector.

[0033] In one or more embodiments, the method of upregulating expression of aquaporin in a plant further comprises: (3) selecting a plant tissue, organ or seed into which the aquaporin gene has been transferred; and (4) regenerating a plant from the plant tissue, organ or seed of step (3).

[0034] In one or more embodiments, the method of downregulating expression or activity of aquaporin in a plant comprises: transferring into the plant an inhibitor of aquaporin gene transcription, protein expression or protein activity.

[0035] In one or more embodiments, the inhibitor comprises an inhibitory molecule that specifically interferes with aquaporin gene transcription and / or expression, or downregulates aquaporin activity.

[0036] In one or more embodiments, the inhibitory molecule targets for inhibition the aquaporin gene or its transcript or expressed protein. Preferably, the inhibitory molecule targets for inhibition the protein set forth in SEQ ID NO: 2 or its RNA counterpart or SEQ ID NO: 1.

[0037] In one or more embodiments, the inhibitory molecule is selected from the group consisting of: (1) a small molecule compound, an antisense nucleic acid, a microRNA, an siRNA, an shRNA, a dsRNA, a sgRNA, a specific antibody or ligand, or a combination thereof, and (2) a nucleic acid construct capable of expressing or forming (1). Preferably, the inhibitory molecule is a dsRNA or a construct thereof that targets for inhibition the aquaporin gene or its transcript.

[0038] In one or more embodiments, the inhibitor is a reagent that knocks out or knocks down the aquaporin gene using a technique selected from the group consisting of ZFN, TALEN and CRISPR, such as sgRNA. In one or more embodiments, the inhibitor further comprises a Cas enzyme (e.g. Cas9), a coding sequence thereof, and / or a nucleic acid construct expressing the Cas enzyme.

[0039] In one or more embodiments, the method of downregulating expression of aquaporin in a plant comprises:

[0040] (i) providing Agrobacterium carrying a nucleic acid construct that can interfere with the expression of a water channel protein gene, said nucleic acid construct containing or producing said inhibitor;

[0041] (ii) contacting a cell or tissue or organ of a plant with the Agrobacterium of step (i), thereby transferring the nucleic acid construct into the plant tissue or organ.

[0042] In one or more embodiments, the nucleic acid construct is an expression vector or an integration vector.

[0043] In one or more embodiments, the method of down-regulating the expression of a water channel protein in a plant further comprises:

[0044] (iii) selecting a plant tissue, organ or seed into which the nucleic acid construct has been transferred; and

[0045] (iv) regenerating a plant from the plant tissue, organ or seed of step (iii).

[0046] The second aspect of the application provides the use of a substance selected from the group consisting of a water channel protein gene or a protein encoded thereby, or a promoter or inhibitor thereof, for modulating the uptake and transport of magnesium ions in a plant, for ameliorating a condition caused by magnesium deficiency or excess in a plant, for modulating the vigour of a plant.

[0047] In one or more embodiments, the water channel protein gene comprises a cDNA sequence, a genomic sequence, or a combination thereof.

[0048] In one or more embodiments, the plant is a Euphorbiales plant; preferably a Euphorbiaceae plant; more preferably a Manihot plant.

[0049] In one or more embodiments, the water channel protein is selected from one or more of the group consisting of PIP, TIP, NIP, SIP, GIP, HIP and XIP.

[0050] In one or more embodiments, the water channel protein is selected from one or more of the group consisting of PIP1, PIP2, PIP3.

[0051] In one or more embodiments, the water channel protein or gene thereof is derived from a Euphorbiales plant, preferably a Euphorbiaceae plant, more preferably a Manihot plant.

[0052] In one or more embodiments, the substance is a water channel protein gene or a protein encoded thereby, or a promoter thereof, and the substance up-regulates the uptake and transport of magnesium ions in a plant, ameliorates a condition caused by magnesium deficiency in a plant, and promotes the growth of a plant.

[0053] In one or more embodiments, the promoter is selected from the group consisting of a small molecule compound, a nucleic acid molecule, or a combination thereof. Preferably, the nucleic acid molecule is a nucleic acid construct containing a water channel protein coding sequence.

[0054] In one or more embodiments, the amino acid sequence of the water channel protein is selected from one or more of the following:

[0055] (a) a polypeptide having the sequence set forth in SEQ ID NO: 1 ;

[0056] (b) a polypeptide derived from (a) by substitution, deletion, or addition of one or more (e.g., 1-20; preferably 1-10; more preferably 1-5) amino acid residues of the sequence set forth in SEQ ID NO: 1, and having the function of the polypeptide of (a); or

[0057] (c) a polypeptide derived from (a) having 90% (preferably 93%; more preferably 95% or 98%) or more homology to the polypeptide sequence of (a), and having the function of the polypeptide of (a).

[0058] In one or more embodiments, the nucleic acid sequence encoding the water channel protein is selected from one or more of the following:

[0059] (1) a polynucleotide encoding a polypeptide as set forth in SEQ ID NO: 1 ;

[0060] (2) a polynucleotide as set forth in SEQ ID NO: 2 or a polynucleotide having 80% (preferably 90%; more preferably 95% or 98%) or more homology thereto;

[0061] (3) a polynucleotide truncated at the 5' end and / or 3' end of the polynucleotide set forth in SEQ ID NO: 2 by 1-60 (preferably 1-30, more preferably 1-10) nucleotides;

[0062] (4) a polynucleotide complementary to any one of the polynucleotides of (1)-(3).

[0063] In one or more embodiments, the substance is an inhibitor of a water channel protein gene or a protein-encoding gene, and the substance down-regulates the uptake and transport of magnesium ions by the plant, ameliorates conditions caused by high magnesium in the plant, and inhibits the growth of the plant.

[0064] In one or more embodiments, the inhibitor comprises an inhibitory molecule that specifically interferes with the transcription and / or expression of a water channel protein gene, or down-regulates the activity of a water channel protein.

[0065] In one or more embodiments, the inhibitory molecule targets the aquaporin gene or its transcript or expressed protein as the inhibition target. Preferably, the inhibitory molecule targets the protein as shown in SEQ ID NO: 2 or its RNA counterpart or SEQ ID NO: 1 as the inhibition target.

[0066] In one or more embodiments, the inhibitory molecule is selected from the group consisting of (1) a small molecule compound, an antisense nucleic acid, a microRNA, an siRNA, an shRNA, a dsRNA, a sgRNA, a specific antibody or a ligand, or a combination thereof, and (2) a nucleic acid construct capable of expressing or forming (1). Preferably, the inhibitory molecule is a dsRNA or a construct thereof targeting the aquaporin gene or its transcript as the inhibition target.

[0067] In one or more embodiments, the inhibitor is a reagent that knocks out or knocks down the aquaporin gene using a technique selected from the group consisting of ZFN, TALEN and CRISPR, such as sgRNA. In one or more embodiments, the inhibitor further comprises a Cas enzyme (such as Cas9), a coding sequence thereof, and / or a nucleic acid construct expressing the Cas enzyme.

[0068] The present application also provides use of an aquaporin gene in identifying a molecular marker for predicting the ability of a plant to absorb and transport magnesium ions, and predicting the growth of a plant.

[0069] In one or more embodiments, the aquaporin gene comprises a cDNA sequence, a genomic sequence, or a combination thereof.

[0070] In one or more embodiments, the plant is a Euphorbiales plant; preferably a Euphorbiaceae plant; more preferably a Manihot plant.

[0071] In one or more embodiments, the aquaporin is selected from one or more of the following: PIP, TIP, NIP, SIP, GIP, HIP and XIP.

[0072] In one or more embodiments, the aquaporin is selected from one or more of the following: PIP1, PIP2, PIP3.

[0073] In one or more embodiments, the aquaporin or its gene is derived from a Euphorbiales plant, preferably a Euphorbiaceae plant, more preferably a Manihot plant.

[0074] In one or more embodiments, the aquaporin has an amino acid sequence selected from one or more of the following:

[0075] (a) a polypeptide having the sequence shown in SEQ ID NO: 1;

[0076] (b) a polypeptide derived from (a) by substitution, deletion, or addition of one or more (e.g., 1-20; preferably 1-10; more preferably 1-5) amino acid residues of the sequence set forth in SEQ ID NO: 1, and which has the function of the polypeptide of (a); or

[0077] (c) a polypeptide derived from (a) having 90% (preferably 93%; more preferably 95% or 98%) or more sequence identity to the polypeptide sequence of (a) and which has the function of the polypeptide of (a).

[0078] In one or more embodiments, the nucleic acid sequence encoding the aquaporin is selected from one or more of the following:

[0079] (1) a polynucleotide encoding a polypeptide as set forth in SEQ ID NO: 1;

[0080] (2) a polynucleotide as set forth in SEQ ID NO: 2 or a polynucleotide having 80% (preferably 90%; more preferably 95% or 98%) or more sequence identity thereto;

[0081] (3) a polynucleotide which is truncated at the 5' end and / or 3' end of the polynucleotide set forth in SEQ ID NO: 2 by 1-60 (preferably 1-30, more preferably 1-10) nucleotides;

[0082] (4) a polynucleotide complementary to the polynucleotide of any one of (1)-(3).

[0083] Another aspect of the present application also provides a polynucleotide or a nucleic acid construct comprising the polynucleotide which inhibits the expression of an aquaporin, the polynucleotide having:

[0084] (1) a sequence set forth in SEQ ID NO: 3 or its corresponding DNA sequence, or a sequence having at least 90% sequence identity thereto,

[0085] (2) an siRNA derived from (1), or

[0086] (3) a structure comprising Formula I:

[0087] Seq 正向 -X-Seq 反向 Formula I,

[0088] in Formula I, Seq 正向 is the sequence set forth in (1) or (2), and Seq 反向 is a polynucleotide which is reverse complementary to Seq 正向 ;

[0089] X is a spacer sequence located between Seq and Seq, and the spacer sequence is complementary to Seq 正向and Seq 反向 Not complementary.

[0090] In one or more embodiments, the siRNA is 10-35 bp in length, preferably 15-30 bp.

[0091] In one or more embodiments, X comprises SEQ ID NO: 4.

[0092] In one or more embodiments, the nucleic acid construct is a vector.

[0093] The positive progress effect of the present application is that it is generally believed that aquaporin has the function of water absorption and transport, and currently no Mg 2+ absorption and transport function has been found. The present application first discovers that aquaporin not only has the water transport function, but also has the Mg 2+ absorption and transport function. BRIEF DESCRIPTION OF DRAWINGS

[0094] Figure 1 : Southern blotting analysis of MePIP2;7 overexpression transgenic cassava. A: Hind III; B: XbaI.

[0095] Figure 2 : Southern blotting analysis of MePIP2;7 RNAi transgenic cassava.

[0096] Figure 3 : Analysis of MePIP2;7 gene expression level in wild type and transgenic cassava.

[0097] Figure 4 : Expression pattern of MePIP2;7 and MGT9 in roots and leaves at different time points under magnesium deficiency.

[0098] Figure 5 : Complementation analysis of bacterial mutant MM281.

[0099] Figure 6 : Yeast two-hybrid analysis.

[0100] Figure 7 : Analysis of BiFC results.

[0101] Figure 8 : Co-localization analysis.

[0102] Figure 9 : Analysis of wild type and transgenic plants under different magnesium ion concentrations. A: Plant photos; B: Root dry weight; C: Aboveground part dry weight.

[0103] Figure 10: Field phenotype of wild type and transgenic plants. A: photos of leaves from different parts of overexpression plants; B: photos of leaves from different parts of interference plants; C: chlorophyll level of basal leaves; D: photos of overexpression plants; E: photos of interference plants; F: weight of storage roots; G: ratio of root length and width.

[0104] Figure 11 : Mg 2+ content analysis in different tissues.

[0105] Figure 12 : Expression analysis of MeMGT9 in different tissues.

[0106] Figure 13 : Iodine staining, starch content and SUT gene expression analysis in different tissues in field. A: iodine staining detection of mature leaves at the base of wild type and transgenic plants (upper: leaves of wild type and overexpression plants; lower: leaves of wild type and RNAi plants). B: starch content in top, middle and basal leaves of wild type and overexpression plants in field. C: starch content in top, middle and basal leaves of wild type and RNAi plants in field. D: starch content in fibrous roots (FR) and developing roots (DR) of wild type and overexpression plants in field. E: starch content in fibrous roots (FR) and developing roots (DR) of wild type and RNAi plants in field. F: expression amount of MeSUT1 in different tissues of wild type and overexpression plants. G: expression amount of MeSUT1 in different tissues of wild type and RNAi plants. H: expression amount of MeSUT2 in different tissues of wild type and overexpression plants. I: expression amount of MeSUT4-1 in different tissues of wild type and overexpression plants. J: expression amount of MeSUT4-1 in different tissues of wild type and RNAi plants. K: expression amount of MeSUT2 in different tissues of wild type and RNAi plants. DETAILED DESCRIPTION

[0107] The present application finds that aquaporin not only has the function of directly transporting water, but also has the function of absorbing and transporting Mg 2+ . Overexpression of aquaporin promotes the absorption and transport of Mg 2+ , and interference inhibits the absorption and transport of Mg 2+ , affecting the growth of plants. The function of aquaporin in absorbing and transporting Mg 2+ is realized by interacting with magnesium ion transporter MGT9.

[0108] Therefore, the present application first provides a method for regulating the absorption and transport of magnesium ions by plants, improving the condition caused by magnesium deficiency or high magnesium in plants, or regulating the growth of plants, comprising: regulating the expression or activity of aquaporin. The plants described herein can be any plant, preferably a model plant (such as Arabidopsis, tobacco) or an Euphorbiaceae plant.

[0109] "magnesium deficiency induced condition" or "low magnesium induced condition" as used herein refers to a change in the plant or the environment caused by the plant absorbing too little magnesium ion. The cause of the magnesium deficiency or low magnesium can be from the environment or from the plant itself, such as a decrease in the concentration of magnesium ion in the environment, or a decrease in the plant's ability to absorb magnesium ion. The magnesium deficiency or low magnesium induced condition includes, but is not limited to, leaf chlorosis, leaf tip and margin discoloration (e.g., from green to light yellow), leaf base discoloration, leaf withering and shedding in severe cases. In addition, the magnesium deficiency or low magnesium can also cause stunted growth, slow growth, abnormal root development, and poor plumping.

[0110] "magnesium deficiency induced condition" or "low magnesium induced condition" as used herein refers to a change in the plant or the environment caused by the plant absorbing too little magnesium ion. The cause of the magnesium deficiency or low magnesium can be from the environment or from the plant itself, such as a decrease in the concentration of magnesium ion in the environment, or a decrease in the plant's ability to absorb magnesium ion. The magnesium deficiency or low magnesium induced condition includes, but is not limited to, leaf chlorosis, leaf tip and margin discoloration (e.g., from green to light yellow), leaf base discoloration, leaf withering and shedding in severe cases. In addition, the magnesium deficiency or low magnesium can also cause stunted growth, slow growth, abnormal root development, and poor plumping. 2+ 、K + "magnesium deficiency induced condition" or "low magnesium induced condition" as used herein refers to a change in the plant or the environment caused by the plant absorbing too little magnesium ion. The cause of the magnesium deficiency or low magnesium can be from the environment or from the plant itself, such as a decrease in the concentration of magnesium ion in the environment, or a decrease in the plant's ability to absorb magnesium ion. The magnesium deficiency or low magnesium induced condition includes, but is not limited to, leaf chlorosis, leaf tip and margin discoloration (e.g., from green to light yellow), leaf base discoloration, leaf withering and shedding in severe cases. In addition, the magnesium deficiency or low magnesium can also cause stunted growth, slow growth, abnormal root development, and poor plumping. 2+ 、K + "magnesium deficiency induced condition" or "low magnesium induced condition" as used herein refers to a change in the plant or the environment caused by the plant absorbing too little magnesium ion. The cause of the magnesium deficiency or low magnesium can be from the environment or from the plant itself, such as a decrease in the concentration of magnesium ion in the environment, or a decrease in the plant's ability to absorb magnesium ion. The magnesium deficiency or low magnesium induced condition includes, but is not limited to, leaf chlorosis, leaf tip and margin discoloration (e.g., from green to light yellow), leaf base discoloration, leaf withering and shedding in severe cases. In addition, the magnesium deficiency or low magnesium can also cause stunted growth, slow growth, abnormal root development, and poor plumping.

[0111] "water channel protein" and "AQP" as used herein interchangeably refer to a polypeptide encoded by a water channel protein gene (including cDNA sequence, genomic sequence, or a combination thereof). The water channel protein includes, but is not limited to, PIP, TIP, NIP, SIP, GIP, HIP, and XIP, wherein PIP includes PIP1, PIP2, or PIP3. Each type of water channel protein as described herein can be derived from any plant, preferably a model plant (e.g., Arabidopsis thaliana, Nicotiana tabacum), or an Euphorbia order plant, such as a Euphorbiaceae plant. In an exemplary embodiment, the water channel protein of cassava MePIP2;7 (GenBank accession No. MN746350, amino acid sequence as shown in SEQ ID NO: 1, coding sequence as shown in SEQ ID NO: 2) is used.

[0112] Variants of polypeptides include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants. Generally, these variants include, but are not limited to, deletions from, insertions into, and / or substitutions of several (usually 1-50, preferably 1-30, 1-20, 1-10, 1-8, 1-5) amino acids, and the addition or deletion of one or several (usually 20 or fewer, preferably 10 or fewer, more preferably 5 or fewer) amino acids at the C-terminus and / or the N-terminus.

[0113] Any polypeptide that is highly homologous to the water channel protein (e.g., 70% or more homologous to the sequence set forth in SEQ ID NO: 1; preferably, 80% or more homologous; more preferably, 90% or more homologous, such as 95%, 98%, or 99% homologous) and has similar or identical function to the protein is also included in the present application. The "identical or similar function" refers primarily to the ability to regulate magnesium ion uptake and transport.

[0114] In the art, conservative substitutions are typically made on the basis of similarity in properties among the amino acids, i.e., conservative variants. In the art, amino acids having similar side chains are often referred to as families, and are well defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), uncharged, nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, lactate, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Also, for example, the addition of one or more amino acids at the amino terminus and / or carboxyl terminus typically does not change the function of the polypeptide or protein. Conservative amino acid substitutions for many commonly known non-genetically encoded amino acids are known in the art. Other conservative substitutions of non-encoded amino acids can be determined based on a comparison of their physical properties to those of genetically encoded amino acids.

[0115] The application also encompasses analogs of the claimed polypeptides. These analogs can differ from the native SEQ ID NO: 1 by differences in the amino acid sequence, by differences in the form of the modification that does not affect the sequence, or by both. These analogs of the proteins include naturally occurring or induced genetic variants. Induced variants can be produced by a variety of techniques, such as random mutagenesis by radiation or exposure to mutagens, by site-directed mutagenesis, or by other known techniques of molecular biology. The analogs also include those having residues other than naturally occurring L-amino acids, for example, D-amino acids, and those having non-naturally occurring or synthetic amino acids such as beta, gamma, etc. It is understood that the proteins of the application are not limited to the representative proteins exemplified above.

[0116] Modifications (which generally do not change the primary structure) include chemical derivatization of the protein in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those modifications that occur during protein synthesis and processing. Such modifications can be accomplished by exposing the protein to enzymes that glycosylate or deglycosylate, such as mammalian glycosylation enzymes. Modified forms also include sequences having phosphorylated amino acid residues, such as phosphotyrosine, phosphoserine, phosphothreonine.

[0117] The polypeptide fragments, derivatives, or analogs of the application can be: (i) polypeptides having one or more conservative or non-conservative amino acid residue substitutions (preferably conservative amino acid residue substitutions), where such substituted amino acid residues can or can not be encoded by the genetic code; or (ii) polypeptides having substituent groups at one or more amino acid residues; or (iii) polypeptides formed by fusing the mature polypeptide to another compound, such as a compound that increases the half-life of the polypeptide, for example, a polyethylene glycol; or (iv) polypeptides formed by adding additional amino acid sequences to the polypeptide, such as leader or secretion sequences, or sequences or proteins for purification of the polypeptide, or prosequences, or fusion proteins. These fragments, derivatives, and analogs are within the scope of those of ordinary skill in the art in view of the definition herein.

[0118] In addition, any biologically active fragment of an aquaporin can be used in the application. As used herein, a biologically active fragment of an aquaporin means a polypeptide that retains all or a portion of the functionality of the full-length aquaporin. Typically, the biologically active fragment retains at least 50% of the activity of the full-length aquaporin. In more preferred embodiments, the active fragment retains 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of the full-length aquaporin.

[0119] The present application also relates to a ligand, an antibody or an antigen-binding fragment thereof of the aquaporin as an inhibitor of the aquaporin. The ligand is an inhibitory ligand; the antibody can be any of a whole antibody, a nanobody, a mono-, bi- or multi-antibody, preferably a mono-antibody. In the present application, the antigen-binding fragment of the antibody is a part of the antibody that can bind to the antigen (aquaporin) and play an inhibitory role to reduce the activity of the antigen (magnesium ion absorption), including but not limited to Fab, F(ab')2, scFv, VHH, etc. The preparation method of the antibody or the antigen-binding fragment thereof is known in the art, for example, by hybridoma or single cell sequencing.

[0120] The present application also relates to a polynucleotide sequence encoding the polypeptide as described herein, including the aquaporin or the variant, the analogue, the derivative thereof, the antibody or the antigen-binding fragment thereof. The polynucleotide can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be the coding strand or the non-coding strand. The coding region sequence encoding the mature polypeptide can be identical to the coding region sequence shown in SEQ ID NO: 2 or a degenerate variant.

[0121] The present application also relates to a variant of the polynucleotide as described above, which encodes a fragment, an analogue and a derivative of the polypeptide having the same amino acid sequence as the present application. The variant of the polynucleotide can be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide, which can be a substitution of one or more nucleotides, a deletion or an insertion, but does not substantially alter the functional properties of the encoded polypeptide. As used herein, a degenerate variant in the present application refers to a nucleic acid sequence that encodes a protein having SEQ ID NO: 1, but differs from the coding region sequence shown in SEQ ID NO: 2. The "polynucleotide encoding a polypeptide" can be a polynucleotide comprising the coding sequence of the polypeptide, or a polynucleotide further comprising additional coding and / or non-coding sequences.

[0122] The present application also relates to polynucleotides that hybridize to the above sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present application particularly relates to polynucleotides that hybridize to the polynucleotides of the present application under stringent conditions. In the present application, "stringent conditions" means: (1) hybridization and washing under low ionic strength and high temperature, such as 0.2xSSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturant, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably 95% or more. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 1.

[0123] It should be understood that although the genes provided in the examples of the present application are derived from cassava, gene sequences of aquaporins derived from other similar plants, especially plants belonging to the same family or genus as cassava, which have certain homology (such as having 70% or more, such as 80%, 85%, 90%, 95%, or even 98% sequence identity) with the sequences of the present application (preferably, the sequence shown in SEQ ID NO: 1) are also included within the scope of the present application, as long as the skilled person can easily isolate the sequence from other plants according to the information provided in the present application after reading the present application. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.

[0124] The full-length sequence of the aquaporin nucleotide of the present application or its fragments can generally be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed according to the nucleotide sequences disclosed in the present application, especially the open reading frame sequences, and a commercially available DNA library or a cDNA library prepared according to conventional methods known to the skilled person can be used as a template for amplification to obtain the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the fragments amplified in each amplification are spliced together in the correct order. Once the relevant sequence is obtained, recombination can be used to obtain the relevant sequence in large quantities. Generally, it is cloned into a vector, then transferred into cells, and then the relevant sequence is isolated from the proliferated host cells by conventional methods. In addition, the relevant sequence can also be synthesized by artificial synthesis. At present, the DNA sequence encoding the protein (or its fragment, or its derivative) of the present application can be obtained completely by chemical synthesis. In addition, mutations can also be introduced into the protein sequence of the present application by chemical synthesis.

[0125] The present application also includes polynucleotide sequences targeting the silencing of aquaporin genes, including but not limited to antisense nucleic acids, microRNAs, siRNAs, shRNAs, dsRNAs, sgRNAs. By studying the aquaporin coding sequences, the inventors provide exemplary dsRNAs (one strand) as shown in SEQ ID NO: 3. The dsRNAs are digested in planta or in vitro by chemical or related enzymes such as (RNase or Dicer) to obtain siRNAs capable of interfering with gene expression. The dsRNAs and / or the siRNAs obtained by digestion thereof can be introduced into plant cells to interfere with gene expression. Thus, the present application includes the dsRNAs and the siRNAs contained therein.

[0126] The present application also provides a recombinant vector comprising the polynucleotide of the present application (e.g. aquaporin coding sequence or dsRNA). As a preferred mode, the recombinant vector comprises a multiple cloning site or at least one restriction site downstream of the promoter. When it is desired to express the gene of interest, the gene of interest is ligated into the appropriate multiple cloning site or restriction site, thereby operably linking the gene of interest to the promoter. As another preferred mode, the recombinant vector comprises (from 5' to 3' direction): a promoter (e.g. 35S promoter), the gene of interest, and a terminator. If desired, the recombinant vector can further comprise an element selected from the group consisting of: a 3' polynucleotidyl signal; an untranslated nucleic acid sequence; a transport and targeting nucleic acid sequence; a resistance selection marker (dihydrofolate reductase, neomycin resistance, hygromycin resistance, and green fluorescent protein, etc.); an enhancer; or an operator. The vector can be an expression vector or an integration vector, the former being used for expressing the gene, dsRNA, related enzymes, sgRNA, etc., and the latter being used for integrating the desired nucleic acid sequence to be expressed into the genome. Exemplary vectors are p1301 and pP35RNAi.

[0127] Methods for preparing recombinant vectors are well known to those of ordinary skill in the art. The expression vector can be a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus, or other vectors. In general, any plasmid and vector can be employed as long as it can replicate and be stable in the host.

[0128] Those of ordinary skill in the art can construct expression vectors containing the genes of the present application using well-known methods. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. When constructing recombinant expression vectors using the genes of the present application, any one of the enhancer, constitutive, tissue-specific, or inducible promoters can be added before the transcription initiation nucleotide.

[0129] The vectors comprising the genes, expression cassettes or of the present application can be used to transform appropriate host cells to allow the host to express the protein. The host cell can be a prokaryotic cell, such as E. coli, Streptomyces, Agrobacterium; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell. Those of ordinary skill in the art will know how to choose the appropriate vector and host cell. Transformation of host cells with recombinant DNA can be performed using conventional techniques known to those of ordinary skill in the art. When the host is a prokaryote (e.g., E. coli), the host can be treated with CaCl2or electroporation can be used. When the host is a eukaryote, DNA transfection methods such as calcium phosphate co-precipitation, conventional mechanical methods (e.g., microinjection, electroporation, liposome packaging, etc.) can be used. Transformation of plants can also be performed using Agrobacterium transformation or biolistics, such as spraying, leaf disc, embryo transformation, floral dip, etc. Transgenic plants can be generated from the transformed plant cells, tissues or organs using conventional methods. The polynucleotide can be expressed in higher eukaryotic cells with enhanced transcription if an enhancer sequence is inserted into the vector. Enhancers are cis-acting elements of DNA that are usually about 10 to 300 base pairs and act on a promoter to increase transcription of a gene.

[0130] Those of ordinary skill in the art will know how to choose the appropriate vector, promoter, enhancer and host cell.

[0131] The polypeptides described herein can be expressed in the cell, on the cell membrane, or secreted outside the cell. If desired, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those of ordinary skill in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic lysis, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0132] The present application provides the use of the aquaporin gene for modulating the uptake and transport of magnesium ions in plants; or for screening substances useful for modulating the uptake of magnesium ions in plants (i.e., the substances modulate the uptake of magnesium ions in plants by modulating the expression of the aquaporin gene); or for identifying the uptake and transport capacity of magnesium ions in plants; or for predicting the molecular marker of the growth of plants. The molecular marker described herein can be a gene, polynucleotide, polypeptide or protein.

[0133] "Modulation" as used herein includes "up-regulation" and "down-regulation". Thus, the methods described herein include up-regulating the expression or activity of a water channel protein, thereby up-regulating the uptake and transport of magnesium ions by a plant, ameliorating a magnesium deficiency condition in a plant, or promoting plant growth. The methods also include down-regulating the expression or activity of a water channel protein, thereby down-regulating the uptake and transport of magnesium ions by a plant, ameliorating a high magnesium condition in a plant, or inhibiting plant growth. The present application also provides the use of a water channel protein gene or encoded protein, or a promoter thereof, for up-regulating the uptake and transport of magnesium ions by a plant, ameliorating a magnesium deficiency condition in a plant, or promoting plant growth; and the use of an inhibitor of a water channel protein gene or encoded protein for down-regulating the uptake and transport of magnesium ions by a plant, ameliorating a high magnesium condition in a plant, or inhibiting plant growth.

[0134] Any substance that increases the activity of a water channel protein, increases its stability, promotes its expression, extends its effective time of action, or promotes its transcription and translation can be used in the present application as a "promoter" of a water channel protein gene for modulating a plant agronomic trait. For example, a vector that increases the transcription, expression, or activity of a water channel protein gene.

[0135] Upon learning of the use of the water channel protein gene, a variety of methods known to those skilled in the art can be used to modulate the expression of the water channel protein gene. For example, an expression unit (e.g., an expression vector or virus) carrying a water channel protein gene can be delivered to a target via a route known to those skilled in the art and allowed to express an active water channel protein; alternatively, a promoter of a water channel protein gene can be contacted with a plant. Illustratively, a method for up-regulating the expression of a water channel protein in a plant includes: (1) providing an Agrobacterium carrying a nucleic acid construct comprising a water channel protein gene, (2) contacting a cell or tissue or organ of a plant with the Agrobacterium of step (1), thereby allowing the nucleic acid construct to be transferred into the plant tissue or organ. Thus, the method further includes: (3) selecting a plant tissue, organ, or seed into which the water channel protein gene has been transferred; and (4) regenerating a plant from the plant tissue, organ, or seed of step (3).

[0136] Alternatively, to up-regulate the activity of a water channel protein, a promoter of a water channel protein can be contacted with a plant. Promoters of a water channel protein include, but are not limited to, agents that promote phosphorylation, hetero-oligomerization, glycosylation, and methylation of a water channel protein, as well as the proton gradient (pH), Ca 2+ and the like, of the cellular environment, as will be known to those skilled in the art.

[0137] In another aspect, any substance that decreases the activity of a water channel protein, decreases its stability, inhibits its expression, decreases its effective time of action, or decreases its transcription and translation can be used in the present application as an inhibitor of a water channel protein. The inhibitor can be used to modulate the uptake and transport of magnesium ions by a plant.

[0138] Accordingly, the present application provides another method of modulating magnesium ion uptake and transport in a plant, the method comprising: reducing expression of a water channel protein gene in the plant (including making the water channel protein gene not expressed or under-expressed); thereby reducing magnesium ion uptake and transport. The expression of the water channel protein gene can be reduced or made absent using a variety of methods well known to those skilled in the art. Illustratively, to down-regulate the water channel protein, an inhibitory molecule that specifically interferes with the transcription and / or expression of the water channel protein gene, or that down-regulates the activity of the water channel protein, can be introduced into the cell or plant, such that the cell or plant does not express or expresses less of the water channel protein. The inhibitory molecule targets the water channel protein gene or its transcript or expressed protein for inhibition. Accordingly, the inhibitory molecule can target the protein as shown in SEQ ID NO: 2 or its RNA counterpart or SEQ ID NO: 1 for inhibition. The inhibitory molecule can be a small molecule compound known to inhibit the activity of the water channel protein, an antibody or a ligand or a binding fragment thereof of the water channel protein, or an antisense nucleic acid, a microRNA, an siRNA, an shRNA, a dsRNA, or an sgRNA that interferes with the expression of the water channel protein gene. In exemplary embodiments, the present application uses a dsRNA or siRNA that targets the nucleotide sequence in positions 57-495 of SEQ ID NO: 2 for inhibition.

[0139] Further, to down-regulate the expression or activity of the water channel protein gene, a gene knockout vector can be introduced into the cell. Accordingly, the inhibitory agent can be an agent that knocks out or knocks down the water channel protein gene using a technology selected from the group consisting of ZFN, TALEN, and CRISPR, such as sgRNA. ZFN, TALEN, and CRISPR / Cas9 technologies suitable for use in the present application are well known in the art. Each of the technologies achieves the knockout of the target gene through the cooperation of a DNA recognition domain and an endonuclease. In these embodiments, the inhibitory agent further comprises a Cas enzyme (e.g., Cas9), a coding sequence thereof, and / or a nucleic acid construct that expresses the Cas enzyme.

[0140] In specific embodiments, the method of down-regulating the expression of the water channel protein in a plant comprises: (i) providing an Agrobacterium carrying a nucleic acid construct that can interfere with the expression of the water channel protein gene, the nucleic acid construct containing or producing the inhibitory agent; (ii) contacting a cell or tissue or organ of the plant with the Agrobacterium of step (i), thereby introducing the nucleic acid construct into the plant tissue or organ; (iii) selecting a plant tissue, organ, or seed into which the nucleic acid construct has been introduced; and (iv) regenerating a plant from the plant tissue, organ, or seed of step (iii).

[0141] Other aspects of the application will be apparent to those of ordinary skill in the art in view of the disclosure herein. The application is further exemplified by the following examples, which should not be construed as limiting. Unless otherwise indicated, the experimental procedures in the following examples were conducted under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

[0142] Examples

[0143] Experimental details

[0144] (I) Experimental materials

[0145] 1. Plant materials

[0146] Wild-type cassava (Manihot esculenta Crantz) TMS60444;

[0147] N. benthamiana.

[0148] 2. Strains

[0149] Escherichia coli: DH5α, TOP10;

[0150] Agrobacterium tumefaciens: LBA4404 (Rifr Chlr), GV3101 (Rifr Tetr);

[0151] Salmonella typhimurium bacterial mutant MM281;

[0152] Yeast NMY51.

[0153] 3. Plasmids

[0154] pMD18-T, pA7-GEMHE, pCAMBIA1301s (pCAMBIA1301 backbone, Kanr), pBSRNAi (pBluescript SK backbone, Ampr), pP35RNAi (pCAMBIA1300 backbone, Kanr), pm-rk, p1300-GFP, p1300-35S-X-cYFP, p1300-35S-nYFP-X, pPR3-N, pBT3-STE, pNubG-Fe65, pTSU2-APP.

[0155] (II) Experimental steps

[0156] 1. Vector construction

[0157] (1) Overexpression vector construction

[0158] Cloning of MePIP2;7 gene with cassava cDNA as template and referring to the sequence on the website (https: / / phytozome.jgi.doe.gov). The PCR product was ligated into pMD18-T, and after sequence verification, it was ligated into pCambia1301S vector through BamH I and Hind III enzyme sites to form the final expression vector p1301-35S::MePIP2;7.

[0159] (2) MePIP2;7 RNA interference vector construction

[0160] Interference fragment amplification: specific primers containing Kpn I and Cla I sites were designed for MePIP2;7 amplification, and the primer sequences are shown in the appendix. The enzyme sites were then changed to Xho I and BamH I to amplify the same fragment as the reverse repeat sequence.

[0161] Intermediate vector construction: the corresponding fragments in pBSRNAi were replaced with target fragments by Kpn I and Cla I, Xho I and BamH I double enzyme digestion. After the first fragment replacement, single colonies were selected by colony PCR for sequence verification, and after the reverse repeat fragment replacement, single colonies were selected by PCR with outer primers or intron upper primers, and the plasmid was extracted for enzyme digestion and PCR verification of the correctness of the constructed hairpin structure.

[0162] Final vector construction: the constructed hairpin structures on the intermediate vector were replaced into pP35RNAi by Kpn I and BamH I double enzyme digestion to form the final RNAi expression vector. All vectors were verified by PCR and enzyme digestion before and after introduction into Agrobacterium tumefaciens.

[0163] (3) Subcellular localization vector construction and transformation

[0164] The CDS sequence of MePIP2;7 without a stop codon was connected to the p1300-GFP vector by Sal I and Spe I double enzyme digestion. The MeMGT9 sequence without a stop codon was connected to the p1300-GFP vector by recombination method, and the correct clone was obtained and transformed into Agrobacterium.

[0165] (4) BiFC and co-localization vector construction and transformation

[0166] MeMGT9 was ligated to p1300-35S-nYFP-X vector by XbaI and BamH I, MePIP2;7 was ligated to p1300-35S-X-cYFP vector by Sal I and Spe I, and Agrobacterium was transformed.

[0167] (5) Construction of yeast two-hybrid vectors

[0168] MeMGT9 was ligated to pm-rk-RFP and pPRN-3 vectors by recombination method, and MePIP2;7 CDS sequence without stop codon was ligated to pBT3-X-STE vector.

[0169] (6) Construction of bacterial mutant vectors

[0170] MePIP2;7, MeMGT4 and MeMGT9 CDS were ligated to pTRC99A vector by homologous recombination method.

[0171] 2. Transformation

[0172] (1) Genetic transformation of cassava suspension callus

[0173] The tissue culture seedlings were cultured for 1 month, and top buds or axillary buds were taken to induce somatic embryos and friable embryogenic calli. The constructed vectors were used for genetic transformation to obtain transgenic plants.

[0174] (2) Tobacco transformation

[0175] p1300-35S-nYFP-MeMGT9 + p1300-35S-cYFP-MePIP2;7, p1300-35S-nYFP-X + p1300-35S-X-cYFP, p1300-GFP, pm-rk-RFP, p1300-MeMGT9-GFP, p1300-MePIP2;7-GFP + CD3-mCherry, p1300-MePIP2;7-GFP + pm-rk-RFP-MeGT9 vectors or combinations were used to transform tobacco respectively.

[0176] (3) Yeast transformation

[0177] pPR3-N + pBT3-MePIP2;7-STE, pNubG-Fe65 + pTSU2-APP, pPR3-N + pTSU2-APP, pPR3-MeMGT4 + pBT3-MePIP2;7-STE, pPR3-MeMGT9 + pBT3-MePIP2;7-STE combinations were used to transform yeast NMY51 respectively.

[0178] (4) Bacterial transformation

[0179] The 1927, pTRC99A, pTRC99A-MePIP2;7, pTRC99A-MeMGT4 and pTRC99A-MeMGT9 vectors were transformed into bacterial mutant MM281, respectively, to obtain positive clones. The positive clone bacterial liquid was collected and spotted on medium containing 10 μM, 100 μM and 1 mM MgSO4, respectively.

[0180] 3. Analysis of growth vigor under different magnesium ion conditions

[0181] The same length of stem segments were cut and planted in MS medium containing 0 μM, 50 μM, 500 μM, 1.5 mM and 6 mM magnesium ions, respectively, at 26°C for 1.5 months, and the growth vigor of the aboveground and underground parts was counted.

[0182] 4. Analysis of magnesium ion content, magnesium ion transporter and starch content in different tissues under field conditions

[0183] The wild type and transgenic strains grown in the field for 6 months were taken as samples, and the samples were kept intact and not damaged. The samples were washed twice with ultrapure water (>18 MΩ cm-1), washed with 25 mM CaCl2(pH 5.0) and ultrapure water again, each for 15 min, and dried at 80°C for 48 h for standby. 15 mg of sample was weighed, 1 ml of 70% HNO3 was added, and the sample was digested in boiling water for 2 h. After cooling, the treated sample was diluted with ddH2O (ultrapure water) to 14 ml, and ICP-MAS (DRC-e, PE Perkin-Elmer Optima3000XL, Applied Biosystems) was used for sample analysis. The standard sample was purchased from PE company. The change of magnesium ion transporter in different tissues was detected by qRT-PCR.

[0184] Analysis of starch content in different tissues at the end of the dark cycle. Total starch extraction and determination method as follows: total starch extraction method: take fresh weight of about 200 mg plant tissue, grind in liquid nitrogen, quickly take 50 mg sample into 2.0 mL EP tube, quickly add 700 μL methanol, shake well, terminate enzyme activity; add 25 μL ribitol stock solution (0.2 mg / mL, polar internal standard, add 25 μL water (conductivity less than 0.05 μs), mix well, check pH value (about 5-6), shake at 70°C for 15 min (open the lid after 1 min), centrifuge at 14,000 g for 3 min; transfer the supernatant to a clean centrifuge tube, add 700 μL pure water, measure the pH value; add 500 μL chloroform (CHCl3) to the precipitate, mix well, shake at 37°C for 5 min, centrifuge at 14,000 g for 3 min, transfer the supernatant to a centrifuge tube, add 500 μL CHCl3 to the mixed supernatant twice, 4,000 rpm for 15 min, take the supernatant, store at 4°C for determination of soluble monosaccharides and total sugars. The remaining precipitate after extraction is washed with methanol 3 times and dried in a 40°C blast drying oven for the next step of starch content determination.

[0185] Starch total content determination method: according to Total Starch kit (K-TSTA, Megazyme), the extracted starch is moistened with 50 μL of 75% ethanol, shaken and mixed well to disperse the sample repeatedly, preventing clumping during heating, 1.5 ml of α-amylase is added, and boiled water is incubated for 12 min to gelatinize the starch in the plant tissue, with shaking and mixing every two minutes to prevent the sample from spitting out of the EP tube due to alcohol evaporation; transfer the EP tube to a 50°C water bath, add 50 μL of starch glucosidase, incubate for 30 min, transfer all the liquid in the EP tube to a 15 mL test tube, make up to 10 mL, take about 100 μL of sample and add 3 mL of GOPOD, incubate at 50°C for 20 min; the blank control uses the same amount of water instead of the sample; the glucose control is 100 μL of glucose standard (1 mg / mL) added to 3 mL of GOPOD reagent. The absorbance of all samples is measured at 510 nm.

[0186] 5. Complementation analysis of bacterial mutant MM281

[0187] Pick the Salmonella typhimurium MM281 monoclonal and inoculate in LB liquid medium containing Chl (34, 136 ul) and Kan (50) and 10 mM Mg 2+ (1 ml mother liquor / 100 mL) at 37°C, shake culture overnight. Wash the collected bacterial solution with sterilized pre-cooled 10% glycerol 4-5 times to prepare MM281 competent cells, and store at -80°C.

[0188] The pTRC99A-MePIP2;7, pTRC99A-MeMGT4, pTRC99A-MeMGT9 vectors were constructed, and then transformed into MM281 competent cells. 1927 was used as a positive control, and MM281-pTrc99A was used as a blank control. Single colonies of MM281, MM281-pTrc99A, MM281-pTrc99A-MeMGT9, MM281-pTrc99A-MeMGT4 and MM281-pTRC99A-MePIP2;7 were picked and cultured in test tubes overnight. Then, the bacteria were inoculated into conical flasks containing corresponding antibiotics at a ratio of 50:1 (50 mL LB:1 mL bacterial solution), and the OD of the bacteria was measured 600 When the OD reached 0.6-0.7, 1 mmol / L IPTG was added for induction, and the induction was continued until the OD reached 1.0. 600 When the OD reached 0.6-0.7, 1 mmol / L IPTG was added for induction, and the induction was continued until the OD reached 1.0. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 mmol / L 5 concentration gradients, and 2 μL of the diluted bacterial solution was spotted on complementary N-Minimal solid medium and cultured at 37°C for 2 days. The growth of the colonies was observed.

[0189] 6. Yeast two-hybrid

[0190] The bait gene MePIP2;7 sequence was amplified and connected to the pBT3-N and pBT3-STE vectors through Sfil enzyme sites to construct the pBT3-N-MePIP2;7 and pBT3-MePIP2;7-STE vectors, in which the N-terminus or C-terminus was fused with VP16-LexA-Cub, respectively. The constructed vectors of different combinations were respectively transformed into the NMY51 yeast strain [MATa his3△200trp1-901 leu2-3,112ade2 LYS2::(lexAop)4-HIS3 ura3::(lexAop)8-lacZ ade2::(lexAop)8-ADE2 GAL4 agar stab], in which pBT3-N-MePIP2;7 and pOst1-NubI were co-transformed, pBT3-MePIP2;7-STE and pOst1-NubI were co-transformed as the experimental group, pTSU2-APP and pNubG-Fe65 were co-transformed as the positive control, and pPR3-N and pTSU2-APP were co-transformed as the negative control. The screening was performed on the two-lack and four-lack media. The analysis showed that the pBT3-MePIP2;7-STE could be used for subsequent interaction verification experiments.

[0191] Construct pPR3-N-MeMGT9 vector, co-transform NMY51 yeast strain with pPR3-N-MeMGT9 and pBT3-MePIP2;7-STE, pBT3-MePIP2;7-STE and pPR3-N, pTSU2-APP and pNubG-Fe65, pPR3-N and pTSU2-APP, and detect whether there is interaction.

[0192] 7. Chlorophyll content determination

[0193] Take the cassava leaves to be tested, rinse them with distilled water, dry them with absorbent paper, take 1 g of leaf mass into a centrifuge tube, and add 5 mL of 95% ethanol to extract chlorophyll. Soak the leaves in 95% alcohol in the dark for 12-24 h to extract pigments until the leaves turn white. Measure OD 663 (nm) and OD 646 (nm) by spectrophotometry; the calculation formula is as follows:

[0194] Chl a = 12.7 x OD 663 - 2.69 x OD 646 ;

[0195] Chl b = 22.9 x OD 646 - 4.68 x OD 663 ;

[0196] Chl a + Chl b = 8.02 x OD 663 + 20.2 x OD 646 .

[0197] The chlorophyll content determined is in units of μg / mL, which needs to be converted to mg / g.

[0198] 8. Leaf iodine staining

[0199] Take leaves at the same field location, remove chlorophyll with 95% alcohol until the leaves turn basically white, then soak them in 1% Lugol's solution (accurately weigh 3.75 g of KI and 1.25 g of I2 into 500 mL of distilled water) at room temperature overnight, wash off the residual iodine solution with water, and observe and take photos.

[0200] 9. Analysis of gene expression by qRT-PCR

[0201] cDNA was synthesized from 1 μg total RNA in a 20 μL reaction system via reverse transcription using ReverTra Ace (product number TRT-101, TOYOBO, Shanghai) for real-time PCR detection. The PCR reaction was performed on a Bio-Rad CFX96 real-time PCR instrument. The 20 μL system included 10 μL of 2×SYBR Master Mix (product number QPK-201, TOYOBO, Shanghai), 50 ng of cDNA, and 400 nmol / L each of forward and reverse primers. The reaction conditions were: 95℃ denaturation for 1 min; 95℃ denaturation for 15 s, 60 s annealing and 30 s extension, for 40-50 cycles; Actin gene was used as an internal control. Each sample was tested in triplicate. The relative expression level of the detected gene was calculated based on the relative Ct value. The formula for calculating the relative expression level of each gene in a single sample is: ΔCt = Ct (Target gene) -Ct (内参) The formula for calculating the relative expression level of a specific gene in two samples is: ΔCt = ΔCt (Sample 1) -ΔCt (Sample 2) .

[0202] Experimental results

[0203] Example 1. Obtaining and Identifying Transgenic Plants

[0204] Figure 1 Southern blotting analysis of MePIP2;7 overexpressing transgenic cassava is shown. The labeling probe is the hygromycin phosphotransferase gene, and the restriction enzyme is Hind III. Figure 1 , A) and Xba I( Figure 1 B), M: λ-HindⅢ. M: marker, 2-23: transgenic lines. OE: overexpression.

[0205] Figure 2 Southern blotting analysis of MePIP2;7 RNA interference in transgenic cassava is shown. The marker probe is the hygromycin phosphotransferase gene. Endonucleases: in the left image, 18 and 27 are digested with Xba I, and the others with HindIII; in the right image, EcoRI digestion is used. M: λ-HindIII. M: marker; 3-27: transgenic lines. Ri: interference.

[0206] Figure 3 This section presents an analysis of MePIP2;7 gene expression levels in wild-type and transgenic cassava. WT: wild-type; Actin: internal control. OE-2, OE-14, OE-23, Ri-7, Ri-18, and Ri-27 were selected for further analysis.

[0207] Example 2. MePIP2;7 interacts with MeGT9

[0208] Figure 4 The expression patterns of MePIP2;7 and MeMGT9 at different time points in roots and leaves under magnesium deficiency conditions were shown. The expression patterns of MePIP2;7 and MeMGT9 under magnesium deficiency conditions were analyzed by qRT-PCR, and the expression of MePIP2;7 and MeMGT9 in roots and leaves was induced, in which MGT9 was up-regulated in leaves, and was first down-regulated, then up-regulated and then down-regulated in roots. MePIP2;7 was first down-regulated, then up-regulated, and then gradually down-regulated in leaves, and was first down-regulated, then gradually up-regulated, and then reached the highest expression at 48 h in roots.

[0209] Figure 5 The results of the bacterial mutant MM281 complementation analysis were shown. 1927: positive control, MM281: mutant, pTRC99A: empty vector, pTRC99A-MePIP2;7, pTRC99A-MeMGT4, pTRC99A-MeMGT9: different vectors transformed MM281 mutant. The bacterial mutant MM281 complementation analysis showed that MePIP2;7 did not have Mg 2+ transporting ability, MeMGT4 and MeMGT9 had Mg 2+ transporting ability.

[0210] Figure 6 The results of the yeast two-hybrid analysis were shown. pPR3-N + pBT3-MePIP2;7: empty vector control, pNubG-Fe65 + pTSU2-APP: positive control, pPR3-N + pTSU2-APP: negative control. Each group of vectors was co-transformed into yeast NMY51. The yeast two-hybrid results showed that MePIP2;7 interacted with MeMGT9, which could grow on the four-lacking plate with 5mM 3-AT and turned blue.

[0211] Figure 7 The results of the BiFC analysis were shown. nYFP + cYFP: negative control, nYFP-MeMGT9 + cYFP-MePIP2;7 co-transformed tobacco. The BiFC results showed that nYFP-MeMGT9 and cYFP-MePIP2;7 co-transformed tobacco, and fluorescence was detected in the plasma membrane, indicating that they interacted.

[0212] Figure 8Co-localization analysis results. p1300-GFP: positive control, pm-rk: positive control (plasma membrane localization), CD3-mCherry: plasma membrane localization marker, p1300-MePIP2;7-GFP, p1300-MeMGT9-GFP and pm-rk-MeMGT9 are different vectors. Subcellular localization results show that MePIP2;7 is localized to the plasma membrane, MeMGT9 is localized to the plasma membrane and chloroplast, and co-localization results show that both can be co-localized to the plasma membrane, indicating that there is some interaction between the two.

[0213] Example 3. MePIP2;7 enhances the absorption of magnesium ions and promotes plant growth under tissue culture conditions

[0214] The same length of stems of wild type and transgenic plants were cut and grafted in MS medium containing 0 uM, 50 uM, 500 uM, 1 mM and 6 mM Mg 2+ , cultured at 26°C (16h light / 8 dark) for 1.5 months, and the differences in growth of wild type and transgenic plants under different conditions were analyzed. The results, as shown in Figure 9 , showed that the interference plants were completely yellow and almost could not grow under complete magnesium deficiency conditions. With the increase of Mg 2+ , the growth of wild type and interference plants became better. Especially under the condition of 1.5 mM Mg 2+ , the average aboveground part of each overexpression plant was 51.29 g, and the average of each interference plant was 25.64 g, indicating that MePIP2;7 could promote plant growth by absorbing more Mg 2+ .

[0215] Example 4. MePIP2;7 promotes the absorption of Mg 2+ under field conditions.

[0216] In May, the stems harvested in Sanya were transplanted to Wuyi farm, and harvested in early November. Overexpression and RNAi transgenic plants were planted in two adjacent rows, and 10 plants of each strain were planted in sequence. Wild type was planted in the middle of each row as a control. The planting density was 80 cm row spacing and 100 cm plant spacing. The results, as shown in Figure 10 , showed that the shape and color of leaves in different parts of wild type and transgenic plants in the same row were analyzed, and it was found that the leaves in different parts of MePIP2;7 overexpression transgenic plants grew normally, and there was no obvious difference compared with wild type. The leaves in different parts of wild type plants in adjacent rows and the upper leaves of RNAi transgenic plants were normal in phenotype, while the lower leaves of RNAi transgenic plants were yellowish and the yellowing phenomenon intensified from top to bottom. The tuberous roots became more elongated, and the weight of storage roots decreased significantly by 74.49% compared with wild type. The storage roots became more elongated, and the length-width ratio was 2.38 times that of wild type.

[0217] Then, the content of Mg 2+ in different tissues of wild type and transgenic plants was analyzed by ICP-MS technology, and the results are shown in Figure 11 . It was found that the content of Mg 2+ in the top leaves of the overexpression plants was slightly higher than that in the wild type, and there was no significant difference in the RNAi plants. In the base mature leaves, the content of Mg 2+ in the wild type was about 8.58 mg / g, the content in the overexpression plants was higher, about 10.632 mg / g, and the content in the RNAi plants was reduced by 16.25%, only 7.18 mg / g. In the developing storage roots, there was no significant difference in the content of Mg 2+ between the wild type and the overexpression plants, which was 1.07 mg / g, while the content in the RNAi plants was 3.15 mg / g, which was 2.94 times of the wild type, indicating that the expression of MePIP2;7 was reduced, resulting in the decrease of the absorption capacity of Mg 2+ .

[0218] In addition, combined with the interaction between MePIP2;7 and MeMGT9, and the Mg 2+ absorption and transport function of MeMGT9, the MeMGT9 in different tissues under field conditions was analyzed by qRT-PCR, and the results are shown in Figure 12 . It was shown that the expression of MeMGT9 in young, middle and base mature leaves in the interference plants was significantly induced. It was verified that the promotion of Mg 2+ absorption and transport by MePIP2;7 was realized by interacting with MeMGT9.

[0219] Iodine staining and starch content analysis showed that the starch accumulated at the end of the dark cycle in the base leaves of the interference plants, and gene expression analysis also verified that the accumulation of carbohydrates in the leaves induced the expression of SUT family genes. The results are shown in Figure 13 : iodine staining of leaves ( Figure 13 A), starch content determination ( Figure 13 B-E), and qRT-PCR analysis of gene expression patterns ( Figure 13 F-K). These results further showed that the interference of MePIP2;7 affected the Mg 2+ absorption and transport function, and confirmed that MePIP2;7 had Mg 2+ absorption and transport function.

[0220] The primers used in this paper are as follows

[0221] Primer name SEQ ID NO: Primer name SEQ ID NO: MePIP2;7 OE FP SEQ ID NO: 5 p1300-nYFP:MeMGT9 FP SEQ ID NO: 25 MePIP2;7 OE RP SEQ ID NO: 6 P1300-nYFP:MeMGT9 RP SEQ ID NO: 26 MePIP2;7 RNAi FP1 forward SEQ ID NO: 7 P1300-MePIP2;7:cYFP FP SEQ ID NO: 27 MePIP2;7 RNAi RP1 forward SEQ ID NO: 8 P1300-MePIP2;7:cYFP RP SEQ ID NO: 28 MePIP2;7 RNAi FP2 reverse SEQ ID NO: 9 pm-rk-MeMGT9 FP SEQ ID NO: 29 MePIP2;7 RNAi RP2 reverse SEQ ID NO: 10 pm-rk-MeMGT9 RP SEQ ID NO: 30 MePIP2;7-GFP FP SEQ ID NO: 11 1300-MeMGT9-GFP FP SEQ ID NO: 31 MePIP2;7-GFP RP SEQ ID NO: 12 1300-MeMGT9-GFP RP SEQ ID NO: 32 HygP-FP SEQ ID NO: 13 RT-MePIP2;7 OE FP SEQ ID NO: 33 HygP-RP SEQ ID NO: 14 RT-MePIP2;7 OE RP SEQ ID NO: 34 pTRC99A-PIP2;7 FP SEQ ID NO: 15 RT-MGT9 FP SEQ ID NO: 35 pTRC99A-PIP2;7 RP SEQ ID NO: 16 RT-MGT9 RP SEQ ID NO: 36 pTRC99A-MGT4 FP SEQ ID NO: 17 RT-MeSUT1 FP SEQ ID NO: 37 pTRC99A-MGT4 RP SEQ ID NO: 18 RT-MeSUT1 RP SEQ ID NO: 38 pTRC99A-MGT9 FP SEQ ID NO: 19 RT-MeSUT2 FP SEQ ID NO: 39 pTRC99A-MGT9 RP SEQ ID NO: 20 RT-MeSUT2 RP SEQ ID NO: 40 pBT3-877-STE FP SEQ ID NO: 21 RT-MeSUT4-1 FP SEQ ID NO: 41 pBT3-877-STE RP SEQ ID NO: 22 RT-MeSUT4-1 RP SEQ ID NO: 42 pPR3-MGT9 FP SEQ ID NO: 23 RT-Meactin FP SEQ ID NO: 43 pPR3-MGT9 RP SEQ ID NO: 24 RT-Meactin RP SEQ ID NO: 44 SEQUENCE LISTING <110> Institute of Molecular Plant Science, Chinese Academy of Sciences <120> Methods of modulating magnesium ion uptake and transport in plants <130> 216326 <160> 44 <170> PatentIn version 3.5 <210> 1 <211> 281 <212> PRT <213> Manihot esculenta <400> 1 Met Ala Lys Glu Val Thr Glu Glu Ala Gly Glu Ala Ser Gln Gln Glu 1 5 10 15 Arg Asp Tyr Val Glu Pro Pro Pro Ala Pro Leu Phe Asp Pro Gln Glu 20 25 30 Leu Gly Leu Trp Ser Phe Tyr Arg Ala Val Ile Ala Glu Phe Ile Ala 35 40 45 Thr Leu Leu Phe Leu Tyr Val Thr Ile Ala Thr Val Ile Gly Tyr Lys 50 55 60 Lys Gln Thr Asp Pro Cys Ala Gly Val Gly Leu Leu Gly Ile Ala Trp 65 70 75 80 Ser Phe Gly Gly Met Ile Phe Ile Leu Val Tyr Cys Thr Ala Gly Ile 85 90 95 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Sequence <220> <223> cDNA <400> 2 atggcgaagg aagtgacaga agaggcagga gaggcttcgc agcaagaaag agactatgta 60 gaaccaccac cagcaccact tttcgaccca caagagcttg gtctctggtc tttttacaga 120 gctgtcatcg cagagttcat cgcaactctt ctctttctct atgtaactat tgccacagtg 180 attggctaca agaagcaaac tgacccttgt gccggcgttg gccttttagg cattgcatgg 240 tcttttggcg gcatgatttt catccttgta tactgtactg ctggaatatc aggtggtcac 300 attaacccag ctgtgacatt gggcttattc ttggcaagga aggtgtcact ggtgcgagct 360 attgcttata tggtggctca gtgtttagga gcaatatgtg gagttggcat agttaagggg 420 attatgaagg atttctataa tgctcaagga ggtggtgcta atactgtggc tgatacatac 480 tctaaaggaa ctgctcttgg agctgagatt attgggactt ttgtccttgt ctacactgtc 540 ttctctgcca ctgatcccaa gcgtaacgca cgtgattctc acgttcctgt tttggctccc 600 ttgccgattg ggtttgcagt ttttatggtg cacttagcca ccatccccat aacaggcact 660 ggaatcaacc cagctcgtag ctttggtgct gctgttatat acaacaacga caaagcctgg 720 gatgaccagt ggatcttctg ggtagggcca tttgtgggag cactagctgc agcaatatat 780 caccagcaca tactgagagc cacagccatt aaagcattgg gatctttcac caccaccacc 840 aactaa 846 <210> 3 <211> 439 <212> DNA <213> Artificial Sequence <220> <223> RNAi <400> 3 tguagaacca ccaccagcac cacuuuucga cccacaagag cuuggucucu ggucuuuuua 60 cagagcuguc aucgcagagu ucaucgcaac ucuucucuuu cucuauguaa cuauugccac 120 agugauuggc uacaagaagc aaacugaccc uugugccggc guuggccuuu uaggcauugc 180 auggucuuuu ggcggcauga uuuucauccu uguauacugu acugcuggaa uaucaggugg 240 ucacauuaac ccagcuguga cauugggcuu auucuuggca aggaaggugu cacuggugcg 300 agcuauugcu uauauggugg cucaguguuu aggagcaaua uguggaguug gcauaguuaa 360 ggggauuaug aaggauuucu auaaugcuca aggagguggu gcuaauacug uggcugauac 420 auacucuaaa ggaacugcu 439 <210> 4 <211> 80 <212> DNA <213> Artificial Sequence <220> <223> RNAi <400> 4 auuuaaauua uuuauuucuu cuuuuccauu uuuuuggcua acauuuucca ugguuuuaug 60 auaucaugca gguacgagcg 80 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 cggatccatg gcgaaggaag tgacagaag 29 <210> 6 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 cccaagcttt 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<223> primer <400> 15 attcgagctc ggtaccatgg cgaaggaagt gacagaattc gagctcggta ccatggcgaa 60 ggaagtgaca ga 72 <210> 16 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 gcaggtcgac tctagattag ttggtggtgg tggtga 36 <210> 17 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 attcgagctc ggtaccatga ggtctcaccc cctgct 36 <210> 18 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 gcaggtcgac tctagattat tccagcaacc gcttgt 36 <210> 19 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 attcgagctc ggtaccatgg tatttcgcga catggc 36 <210> 20 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 gcaggtcgac tctagatcaa gatccaacca gacctt 36 <210> 21 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 tctattttat gtaatggcca ttacggccat ggcgaaggaa gtgacaga 48 <210> 22 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 cgaattcctg cagatggccg aggcggcccc gttggtggtg gtggtga 47 <210> 23 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 gtatcaacgc agagtggcca ttacggccat ggtatttcgc gacatggc 48 <210> 24 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 atcgaattct cgagaggccg aggcggcctc aagatccaac cagacctt 48 <210> 25 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 cacaacgtct atatcatggc tctagacatg gtatttcgcg acatggc 47 <210> 26 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 26 cgagctctat cgatcaatca ggatcctcaa gatccaacca gacctttgt 49 <210> 27 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 27 accatcacca tcacgccatg gtcgacatgg cgaaggaagt gacaga 46 <210> 28 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 28 cgttcttctg cttgtccatc actagtacgt tggtggtggt ggtgaaag 48 <210> 29 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 29 cggagctagc tctagaatgg tatttcgcga catggc 36 <210> 30 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 30 tgctcaccat ggatccagat ccaaccagac ctttgt 36 <210> 31 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 31 accatcacgc catggtcgac atggtatttc gcgacatggc 40 <210> 32 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 32 gctcaccatc actagtacag atccaaccag acctt 35 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 33 cgattgggtt tgcagttttt 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 34 tggctgtggc tctcagtatg 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 35 tttctagctg cacgcacaac 20 <210> 36 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 36 cgagcagtca accttgtcat tg 22 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 37 attccttttg caatggcatc 20 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 38 tttggctgag gggatatcag 20 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 39 caaaatggcc ttgagctttc 20 <210> 40 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 40 gctccaggtc catcaccta 19 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 41 tgtagagcgc ttcttgctga 20 <210> 42 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 42 gcagacttga gattggcaca 20 <210> 43 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 43 tgatgagtct ggtccatcca 20 <210> 44 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 44 cctcctacga cccaatctca 20

Claims

1. A method of modulating the uptake and translocation of magnesium ions by a plant, ameliorating a condition caused by magnesium deficiency or excess in a plant, or modulating the growth habit of a plant, the plant being cassava, comprising: modulating the expression or activity of a water channel protein having an amino acid sequence as set forth in SEQ ID NO:

1.

2. The method of claim 1, wherein, the method comprises up-regulating the expression or activity of a water channel protein, thereby up-regulating the uptake and transport of magnesium ions by the plant, ameliorating a magnesium deficiency condition in the plant, or promoting plant growth, or the method comprises down-regulating the expression or activity of a water channel protein, thereby down-regulating the uptake and transport of magnesium ions by the plant, ameliorating a high magnesium condition in the plant.

3. The method as described in claim 2, characterized in that, the up-regulating the expression or activity of a water channel protein in the plant comprises: (1) introducing a water channel protein gene into the plant to obtain a transformed plant; and / or (2) contacting a water channel protein gene or an agent that promotes the protein encoded by the gene with the plant.

4. The method of claim 3, wherein, The facilitator includes an agent that phosphorylates, heteromerizes, glycosylates and methylates aquaporins or an agent that increases the proton gradient, Ca 2+ of the cellular environment.

5. The method of claim 2, wherein, the down-regulating the expression or activity of a water channel protein comprises introducing an inhibitor of water channel protein gene transcription, protein expression or protein activity into the plant.

6. The method of claim 5, wherein, the inhibitor comprises an inhibitory molecule that specifically interferes with water channel protein gene transcription and / or expression, or down-regulates water channel protein activity.

7. The method of claim 6, wherein, the inhibitory molecule targets the water channel protein gene or its transcript or expressed protein for inhibition.

8. The method of claim 7, wherein, the inhibitory molecule targets a protein having an amino acid sequence as set forth in SEQ ID NO: 1 or an RNA counterpart thereof for inhibition.

9. The method of claim 6, wherein, the inhibitory molecule is selected from the group consisting of: (1) a small molecule compound, an antisense nucleic acid, a microRNA, a specific antibody or ligand, an agent that knocks out or knocks down the water channel protein gene using a technique selected from the group consisting of ZFN, TALEN and CRISPR, or a combination thereof, and (2) a nucleic acid construct that expresses or forms (1).

10. The method of claim 9, wherein, the inhibitory molecule is selected from the group consisting of siRNA, shRNA, dsRNA and sgRNA.

11. The method of any one of claims 1-10, wherein, the nucleic acid sequence encoding the water channel protein is selected from one or more of: (1) a polynucleotide that encodes a polypeptide as set forth in SEQ ID NO: 1; (2) a polynucleotide as set forth in SEQ ID NO: 2; (3) a polynucleotide that is complementary to the polynucleotide of any one of (1)-(2).

12. Use of a substance selected from the group consisting of a water channel protein gene or encoded protein, or an agent that promotes or inhibits the same, in modulating the uptake and transport of magnesium ions by a plant, ameliorating a magnesium deficiency or high magnesium condition in the plant, or modulating the growth of the plant, the plant being cassava, the water channel protein having an amino acid sequence as set forth in SEQ ID NO:

1.

13. The use according to claim 12, characterized in that, the substance is a water channel protein gene or encoded protein, or an agent that promotes the same, and the substance up-regulates the uptake and transport of magnesium ions by the plant, ameliorates a magnesium deficiency condition in the plant, or promotes plant growth.

14. The use according to claim 12, characterized in that, the agent is selected from the group consisting of a small molecule compound, a nucleic acid construct that contains a water channel protein coding sequence, or a combination thereof.

15. The use according to claim 12, characterized in that, The facilitators include agents that phosphorylate, oligomerize, glycosylate and methylate aquaporins or agents that increase the proton gradient, Ca 2+ of the cellular environment.

16. The use of claim 12, wherein, the substance is an inhibitor of a water channel protein gene or encoded protein, and the substance down-regulates the uptake and transport of magnesium ions by the plant, ameliorates a high magnesium condition in the plant.

17. The use of claim 12, wherein, the inhibitor comprises an inhibitory molecule that specifically interferes with water channel protein gene transcription and / or expression, or down-regulates water channel protein activity.

18. The use according to claim 17, characterized in that, The inhibitory molecule targets for inhibition the aquaporin gene or its transcript or expressed protein.

19. The use of claim 17, wherein, The inhibitory molecule targets for inhibition the protein as set forth in SEQ ID NO: 2 or its RNA counterpart or SEQ ID NO:

1.

20. The use of claim 17, wherein, The inhibitory molecule is selected from the group consisting of (1) a small molecule compound, an antisense nucleic acid, a microRNA, a specific antibody or ligand, a reagent that knocks out or knocks down the aquaporin gene using a technology selected from the group consisting of ZFN, TALEN and CRISPR, or a combination thereof, (2) a nucleic acid construct that is capable of expressing or forming (1).

21. The use according to claim 20, characterized in that, The inhibitory molecule is selected from the group consisting of siRNA, shRNA, dsRNA and sgRNA.

22. The use according to any one of claims 12 to 21, characterized in that, The nucleic acid sequence encoding the aquaporin protein is SEQ ID NO:

2.

23. Use of an aquaporin gene in a molecular marker for identifying the ability of a plant, which is cassava, to absorb and transport magnesium ions, for predicting the vigor of the plant, the amino acid sequence of the aquaporin protein being as set forth in SEQ ID NO:

1.

24. Use as claimed in claim 23, characterised in that, The nucleic acid sequence encoding the aquaporin protein is as set forth in SEQ ID NO: 2.

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