Application of KR2 and KR3 proteins and coding genes thereof in efficient utilization of corn potassium

Abstract

The invention discloses application of KR2 and KR3 proteins and coding genes thereof in efficient utilization of corn potassium, the corn KR2 and KR3 genes are knocked out by utilizing a CRISPR / Cas9 gene editing technology, a mutant plant with high potassium absorption and utilization efficiency is obtained, under the low-potassium condition, the leaf persistence of the mutant is stronger, the biomass and potassium content of the overground part of the mutant plant are higher, and the corn potassium content is higher. The KR2 and KR3 genes of the corn are knocked out in the corn, the phenotype of the KR2 and KR3 genes of the corn is larger than that of a wild plant, it is proved that after the KR2 and KR3 genes of the corn are knocked out in the corn, the potassium absorption and utilization efficiency of the corn can be improved, the potassium content of corn leaves can be regulated and controlled, biomass is improved, and gene resources are provided for cultivating new varieties. Compared with a traditional breeding mode, the breeding method for efficient absorption and utilization of potassium has the advantages that the breeding time is short, the purposiveness is high, the breeding period is remarkably shortened, the breeding efficiency is improved, the breeding method has great application value in crop molecule improvement, and a theoretical basis is provided for clarification of a molecular mechanism of potassium nutrition absorption and utilization of plants.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of KR2 and KR3 proteins and encoding genes thereof in efficient utilization of corn potassium. Background Art

[0002] Potassium is one of the three major mineral elements necessary for plant growth and development. In plants, potassium ions are the most abundant cations in plants and play an important role in plant signal transduction and stress response. Potassium is extremely important for maintaining enzyme activity. It is an activator of enzymatic reactions, regulates the osmotic potential of cells, enhances plant stress resistance, and affects photosynthesis and crop yield and quality.

[0003] Potassium is essential for the growth, development, yield and quality of crops. When there is a serious shortage of potassium fertilizer in the field, plant growth slows or stagnates, stems become weak, yield and quality decrease, and disease resistance weakens.

[0004] In recent years, corn has become the crop with the highest yield in my country. Corn cultivation requires a large amount of potassium fertilizer, but most of my country's arable land is potassium deficient. Improving soil fertility by applying potassium fertilizer will increase agricultural production costs, and the use of chemical fertilizers will also have an impact on the environment.

[0005] Potassium content in the soil is unstable, and the way and efficiency of plant absorption and utilization of potassium vary. Discovering the key genes for efficient absorption and utilization of potassium in corn, analyzing the molecular genetic mechanism of corn potassium efficiency traits, and providing theoretical and technical support for genetic improvement of corn potassium nutritional efficiency are of great significance for achieving high and stable corn yields. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention aims to provide an application of KR2 and KR3 proteins and their encoding genes in the efficient utilization of potassium in corn, and the technical problem solved is to improve the potassium absorption capacity of plants, reduce the demand for potassium elements in plants, improve the potassium utilization efficiency of plants, increase the potassium content in plants, regulate the potassium ion balance of plants, regulate the potassium ion content of plant leaves, and cultivate high-yield crops; by knocking out the KR2 and KR3 genes, mutant plants with high potassium absorption and utilization efficiency are obtained. Under normal culture conditions, there is no obvious difference between the mutant plants and wild-type plants. Under low-potassium conditions, the third leaves of the mutants are more green, and the biomass and potassium content of the aboveground parts of the mutant plants are higher, showing a larger phenotype than the wild-type plants, which confirms that after the corn KR2 and KR3 genes are knocked out in corn, the potassium absorption and utilization efficiency of corn can be improved and the potassium content of corn leaves can be regulated, thereby improving the tolerance of corn under low-potassium conditions and improving the potassium absorption and utilization efficiency, and ultimately achieving an increase in biomass.

[0007] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0008] Application of KR2 and KR3 proteins or their encoding genes or biological materials containing their encoding genes in any of the following aspects:

[0009] P1) Application in increasing potassium ion content in plant leaves;

[0010] P2) Application in regulating potassium ion balance in plants;

[0011] P3) Application in increasing plant biomass;

[0012] P4) Application in the breeding of plants with high potassium utilization efficiency;

[0013] The amino acid sequence of the KR2 protein is any one of the following:

[0014] A1) the sequence shown in SEQ ID NO.1;

[0015] A2) a protein sequence having the same biological function obtained by replacing and / or deleting and / or adding one or more amino acid residues in the sequence shown in SEQ ID NO.1;

[0016] A3) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1) or A2);

[0017] The amino acid sequence of the KR3 protein is any one of the following:

[0018] A4) the sequence shown in SEQ ID NO.4;

[0019] A5) a protein sequence having the same biological function obtained by replacing and / or deleting and / or adding one or more amino acid residues in the sequence shown in SEQ ID NO.4;

[0020] A6) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A4) or A5).

[0021] The above-mentioned protein may be derived from corn.

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

[0023] In the above proteins, the protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The protein tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0024] The plant mentioned above may be any of the following:

[0025] 1) Dicotyledonous plants;

[0026] 2) Monocots,

[0027] 3) Gramineae plants,

[0028] 4) Gramineae,

[0029] 5) Zea mays;

[0030] 6) Corn.

[0031] Preferably, the nucleotide sequence of the gene encoding the KR2 protein is any one of the following:

[0032] B1) the nucleotide sequence shown in SEQ ID NO.2;

[0033] B2) the nucleotide sequence shown in SEQ ID NO.3;

[0034] B3) a nucleotide sequence in which one or more nucleotides are replaced, deleted and / or added as the nucleotide sequence shown in SEQ ID NO.2 and the nucleotide sequence expresses a protein with the same function;

[0035] The nucleotide sequence of the gene encoding the KR3 protein is any one of the following:

[0036] B4) the nucleotide sequence shown in SEQ ID NO.2;

[0037] B5) the nucleotide sequence shown in SEQ ID NO.3;

[0038] B6) A nucleotide sequence in which one or more nucleotides are replaced, deleted and / or added to the nucleotide sequence shown in SEQ ID NO. 2 and the nucleotide sequence expresses a protein with the same function.

[0039] Preferably, the biological material is any one of the following C1) to C9):

[0040] C1) a nucleic acid molecule encoding the KR2 protein and the KR3 protein according to claim 1;

[0041] C2) an expression cassette containing the nucleic acid molecule described in C1);

[0042] C3) a recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2);

[0043] C4) a recombinant microorganism containing the nucleic acid molecule described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3);

[0044] C5) a transgenic plant cell line containing the nucleic acid molecule described in C1), or a transgenic plant cell line containing the expression cassette described in C2), or a transgenic plant cell line containing the recombinant vector described in C3);

[0045] C6) transgenic plant tissue containing the nucleic acid molecule described in C1), or transgenic plant tissue containing the expression cassette described in C2), or transgenic plant tissue containing the recombinant vector described in C3);

[0046] C7) a transgenic plant organ containing the nucleic acid molecule described in C1), or a transgenic plant organ containing the expression cassette described in C2), or a transgenic plant organ containing the recombinant vector described in C3);

[0047] C8) a nucleic acid molecule that inhibits or reduces the expression of a gene encoding the protein of claim 1 or a nucleic acid molecule that inhibits or reduces the activity of the protein of claim 1;

[0048] C9) An expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing the nucleic acid molecule described in C8).

[0049] Any of the above-mentioned nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA.

[0050] Any of the above-mentioned nucleic acid molecules can be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0051] Any of the above-mentioned expression cassettes may include a promoter, the nucleic acid molecule described in C1) or C2) above, and a terminator. Promoters that can be used in the present invention include, but are not limited to, constitutive promoters, tissue, organ and development-specific promoters, and inducible promoters. Furthermore, the expression cassette may also include an enhancer sequence.

[0052] Any of the above-mentioned vectors refers to a vector that can carry the nucleic acid molecule described in C1) or C2) into the host cell for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmid, phage (such as λ phage or M13 filamentous phage, etc.), cosmid (i.e., cosmid), Ti plasmid, viral vector (such as retrovirus (including lentivirus), adenovirus, adeno-associated virus, etc.).

[0053] Any of the above-mentioned recombinant vectors refers to a recombinant DNA molecule constructed by connecting the nucleic acid molecule described in C1) or C2) with the vector in vitro. The recombinant vector containing the nucleic acid molecule described in C1) or C2) can be constructed using an existing plant expression vector. The plant expression vector includes a binary Agrobacterium vector and a vector that can be used for plant microprojectile bombardment, etc. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Company), etc. The plant expression vector may also contain the 3′ non-translated region of the foreign gene, i.e., a polyadenylic acid signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide the addition of polyadenylic acid to the 3' end of the mRNA precursor, such as the non-translated region transcribed at the 3' end of the Agrobacterium crown gall induction (Ti) plasmid gene (such as the nopaline synthase gene Nos) and the plant gene (such as the soybean storage protein gene) all have similar functions. When using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive and can be natural or synthetic. The translation start region can come from the transcription start region or the structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic marker genes (such as nptII gene that confers resistance to kanamycin and related antibiotics, bar gene that confers resistance to the herbicide phosphinothricin, hph gene that confers resistance to the antibiotic hygromycin, dhfr gene that confers resistance to methotrexate, EPSPS gene that confers resistance to glyphosate) or chemical resistance marker genes (such as herbicide resistance genes), mannose-6-phosphate isomerase gene that provides the ability to metabolize mannose. Considering the safety of transgenic plants, no selective marker gene can be added, and transformed plants can be directly screened by adversity.

[0054] Any of the above-mentioned microorganisms may be bacteria, fungi, actinomycetes, protozoa, algae or viruses. Wherein, the bacteria may be from Escherichia sp., Erwinia sp., Agrobacterium sp., Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., Bacillus sp., etc., but not limited thereto, for example, the bacteria may be Escherichia coli, Bacillus subtilis or Bacillus pumilus. The fungus may be yeast, and the yeast may be from the genus Saccharomyces (such as Saccharomyces cerevisiae), the genus Kluyveromyces (such as Kluyveromyces lactis), the genus Pichia (such as Pichia pastoris), the genus Schizosaccharomyces (such as Schizosaccharomyces pombe), the genus Hansenula (such as Hansenula polymorpha), etc., but not limited thereto. The fungus may also be from the genus Fusarium (Fusarium sp.), the genus Rhizoctonia (Rhizoctonia sp.), the genus Verticillium (Verticillium sp.), the genus Penicillium (Penicillium sp.), the genus Aspergillus (Aspergillus sp.), the genus Cephalosporium (Cephalosporium sp.), etc., but not limited thereto. The actinomycetes may be from Streptomyces sp., Nocardia sp., Micromonospora sp., Streptosporangium sp., Actinoplanes sp., Thermoactinomyces sp., etc., but not limited thereto. The algae may be from Fucus sp., Achnanthes sp., Amphiprora sp., Amphora sp., Ankistrodesmus sp., Asteromonas sp., Boekelovia sp., etc., but not limited thereto. The virus may be rotavirus, herpes virus, influenza virus, adenovirus, etc., but not limited thereto.

[0055] Any of the above-mentioned recombinant microorganisms refers to a recombinant microorganism with a changed function obtained by operating and modifying the genes of the target microorganism. For example, a recombinant microorganism obtained by introducing the above-mentioned recombinant vector into the target microorganism. The recombinant microorganism can be understood to refer not only to a specific recombinant microorganism, but also to the offspring of such a cell, and due to natural, accidental or intentional mutations and / or changes, the offspring may not be completely identical to the original parent cell, but is still included in the scope of the recombinant microorganism.

[0056] Preferably, the above application is achieved by inhibiting or reducing the expression level and / or activity of KR2 protein and KR3 protein.

[0057] The said substances which inhibit or reduce the activities of KR2 protein and KR3 protein are proteins, polypeptides or small molecule compounds which inhibit or reduce the functions of KR2 protein and KR3 protein.

[0058] The substance that reduces the content of KR2 protein and KR3 protein can be a substance that inhibits the synthesis of KR2 protein and KR3 protein or promotes the degradation of KR2 protein and KR3 protein or knocks down (knockdown) or knocks out the genes encoding KR2 protein and KR3 protein.

[0059] A method for improving plant potassium utilization efficiency, wherein the expression amount and / or activity of KR2 protein and KR3 protein are inhibited or reduced by transgenic technology to obtain mutant plants, and the plant potassium utilization efficiency is improved;

[0060] The amino acid sequence of the KR2 protein is any one of the following:

[0061] A1) the sequence shown in SEQ ID NO.1;

[0062] A2) a protein sequence having the same biological function obtained by replacing and / or deleting and / or adding one or more amino acid residues in the sequence shown in SEQ ID NO.1;

[0063] A3) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1) or A2);

[0064] The amino acid sequence of the KR3 protein is any one of the following:

[0065] A4) the sequence shown in SEQ ID NO.4;

[0066] A5) a protein sequence having the same biological function obtained by replacing and / or deleting and / or adding one or more amino acid residues in the sequence shown in SEQ ID NO.4;

[0067] A6) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A4) or A5).

[0068] A method for increasing plant biomass, wherein the expression amount and / or activity of KR2 protein and KR3 protein are inhibited or reduced by transgenic technology to obtain mutant plants, and the plant biomass is increased;

[0069] The amino acid sequence of the KR2 protein is any one of the following:

[0070] A1) the sequence shown in SEQ ID NO.1;

[0071] A2) a protein sequence having the same biological function obtained by replacing and / or deleting and / or adding one or more amino acid residues in the sequence shown in SEQ ID NO.1;

[0072] A3) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1) or A2);

[0073] The amino acid sequence of the KR3 protein is any one of the following:

[0074] A4) the sequence shown in SEQ ID NO.4;

[0075] A5) a protein sequence having the same biological function obtained by replacing and / or deleting and / or adding one or more amino acid residues in the sequence shown in SEQ ID NO.4;

[0076] A6) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A4) or A5).

[0077] Preferably, the coding genes of KR2 protein and KR3 protein are edited in corn by CRISPR / Cas9 technology to inhibit the expression level and / or activity of KR2 protein and KR3 protein in corn; the gRNA target sequence is shown in SEQ ID NO.7.

[0078] A KR2 and KR3 gene mutant material, wherein the mutant material is any one of the following:

[0079] 1) The KR2 gene sequence shown in SEQ ID NO.2 lacks two bases CA at positions 197 and 198;

[0080] The KR3 gene sequence shown in SEQ ID NO.5 has a missing base T at position 115;

[0081] or 2) the nucleotide sequence of the gene of the kr2 / 3 mutant material is:

[0082] The KR2 gene sequence shown in SEQ ID NO.2 lacks two bases TG at positions 199 and 200;

[0083] The 114th position of the KR3 gene sequence shown in SEQ ID NO.5 is missing a base A.

[0084] The substance for knocking down (knocking down) the KR2 and KR3 protein encoding genes can be any substance that can inhibit the expression of the above-mentioned KR2 and KR3 protein encoding genes, such as gRNA (such as sgRNA), mRNA, siRNA, dsRNA, shRNA, miRNA, antisense RNA, etc.

[0085] The substance for knocking out the KR2 and KR3 protein coding genes can be a substance that achieves the host cell not producing the functional protein products of the KR2 and KR3 genes in any way, such as removing all or part of the coding gene sequence, introducing mutations so that no functional protein is produced, removing or changing regulatory components (such as promoter editing) so that the coding gene sequence is not transcribed, preventing translation by binding to mRNA, etc. Usually, the knockout is performed at the genomic DNA level, so that the offspring of the cell also permanently carry the knockout.

[0086] The substance for knocking out the KR2 and KR3 protein encoding genes may be a substance that causes the KR2 and KR3 genes in the plant to mutate (the mutation may be a deletion mutation and / or an insertion mutation and / or a base substitution) thereby losing their activity. The mutation method may be any method known in the technical field, such as the zinc finger protein ZFN gene editing system, the TALENs gene editing system, the CRISPR / Cas9 gene editing system, T-DNA insertion, etc.

[0087] The beneficial effects of the present invention are:

[0088] The present invention finds that KR2 and KR3 proteins can regulate the efficiency of plant potassium absorption and utilization, providing new gene targets and gene resources for cultivating new varieties with efficient potassium utilization.

[0089] Compared with traditional breeding methods, the breeding method for efficient potassium absorption and utilization of the present invention has the advantages of short breeding time and strong purpose, significantly shortens the breeding cycle, improves breeding efficiency, and has great application value for cultivating potassium-efficient crops.

[0090] The present invention has important theoretical and practical significance in reducing the demand of plants for potassium, improving the potassium absorption capacity of plants, improving the potassium utilization efficiency of plants, increasing the potassium content in plants, regulating the potassium ion balance of plants, and regulating the potassium ion content in plant leaves. It also provides a theoretical basis for clarifying the molecular mechanism of plant potassium nutrient absorption and utilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0092] Figure 1 The results of KR2 and KR3 gene structure and mutation sequence detection in kr2 / 3 mutant plants.

[0093] Figure 2 This is the phylogenetic relationship of the family of two proteins KR2 and KR3; among them, KR2 and KR3 have the highest homology.

[0094] Figure 3 Phenotypic observation of kr2 / 3 mutant material in hydroponic culture. A: kr2 / 3 mutant material in low potassium (LK, 0.03 mM K + ) condition, photos of the whole plant and photos of the second to fourth leaves. B: Phenotypic observation of kr2 / 3 mutant material under normal potassium (HK, 1.85 mM K + ) conditions, photos of the whole plant and the second to fourth leaves; scale bar is 10 cm.

[0095] Figure 4 Results of determination of dry weight and potassium content of the third leaf of hydroponic kr2 / 3 mutant materials. A: Dry weight of the third leaf under HK (1.85 mM) and LK (0.03 mM) treatments. B: Potassium content of the third leaf under HK and LK treatments. Data are shown as mean ± error, n = 3 (biological replicates), one-way analysis of variance (ANOVA, LSD method). DETAILED DESCRIPTION

[0096] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the following embodiments are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0097] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0098] The quantitative experiments in the following examples were all repeated three times, and the results were averaged.

[0099] The pBUE411 vector in the following example is recorded in the document “Wang Xing, Fang et al. ACRISPR / Cas9 toolkit for multiplex genome editing in plants Maize ZmPT7 regulates Pi uptake and redistribution which is modulated by phosphorylation. Plant biotechnology journal BMC Plant Biology 2014, 14: 327 vol. 18, 12 (2020): 2406-2419.”.

[0100] Agrobacterium tumefaciens EHA105 in the following examples is described in the document “Nyaboga et al. Agrobacterium-mediated genetic transformation of yam (Dioscorea arotunda): an important tool for functional study of genes and crop improvement. Frontiers in Plant Science 2014, 5: 463.”

[0101] The maize inbred line used in the embodiment of the present invention is ND101 (PI 612589), and the germplasm information can be queried at GRIN-Global https: / / npgsweb.ars-grin.gov / gringlobal / search.

[0102] The KR2 and KR3 genes in the following embodiments can be queried on the MaizeGDB website (https: / / www.maizegdb.org / ). The gene number of the V3 version of KR2 is: GRMZM2G143205, and the gene number of the V4 version is Zm00001d003958; the gene number of the V3 version of KR3 is: GRMZM2G152661, and the gene number of the V4 version is Zm00001d025235. In corn ND101, the amino acid sequence of the protein encoded by the KR2 gene is shown in SEQ ID NO.1, which consists of 996 amino acid residues; the CDS sequence of the KR2 gene is shown in SEQ ID NO.2, which consists of 2991 bases; the nucleotide sequence of the KR2 gene is shown in SEQ ID NO.3, which consists of 8444 bases, contains 12 exons, and has a structure as shown in Figure 1The amino acid sequence of the protein encoded by the KR3 gene is shown in SEQ ID NO.4, which consists of 865 amino acid residues; the CDS sequence of the KR3 gene is shown in SEQ ID NO.5, which consists of 2598 bases; the nucleotide sequence of the KR3 gene is shown in SEQ ID NO.6, which consists of 7468 bases, contains 13 exons, and has a structure as shown in Figure 1 shown.

[0103] The corn material in the embodiment of the present invention is cultivated in a hydroponic manner:

[0104] In the hydroponic system, the seeds were sown in vermiculite at 28°C for 5 days until they had one leaf and one heart. The endosperm was removed and the seedlings were grown in HK nutrient solution for 2 days. HK (1.85 mM K + ) and LK (0.03 mM K + ) treatment. The formula of hydroponic nutrient solution is as follows:

[0105] Table 1. HK hydroponic nutrient solution formula

[0106]

[0107] Table 2. LK hydroponic nutrient solution formula

[0108]

[0109]

[0110] Note: The contents shown in the table are working solution concentrations.

[0111] In addition, a trace solution of 2000× (working solution concentration: 1 μmol / LMnSO) needs to be added to the nutrient solution. 4 , 1μmol / LZnSO 4 7H 2 O, 0.01 μmol / L CuSO 4 ·5H 2 O, 0.005 μmol / L (NH 4 ) 6 Mo 7 O 24 ·4H 2 O and 1 μmol / L H 3 BO 3 ): 0.5mL / kg soil; iron salt 100× (working solution concentration: 0.1mmol / LFe-EDTA). Finally, use NaOH to adjust the pH to 5.8-6.0. Use a 20L hydroponic box to culture corn in a 28℃ culture room. One hydroponic box cultured 12 seedlings, and the hydroponic solution was changed every 2 days. Phenotypic observation was performed 14-16 days after treatment.

[0112] Example 1 Construction and detection of maize kr2 / 3 mutant material

[0113] In order to study the functions of KR2 and KR3 genes and their encoded proteins in corn, the present invention uses CRISPR / Cas9 technology to edit the KR2 and KR3 genes in the corn genome to obtain kr2 / 3 mutants.

[0114] The specific steps are as follows:

[0115] 1. Construction and testing of KR2 and KR3 CRISPR / Cas9 gene editing vectors

[0116] 1. Design of gRNA target sequence

[0117] The present invention designs a target site for KR2 and KR3 genomic DNA, which simultaneously targets the exons of two genes. The target site sequence of sgRNA is as follows: 5'-GAGAGATGGTCACACATGG-3' (SEQ ID NO. 7).

[0118] 2. Preparation of KR2 and KR3 CRISPR / Cas9 gene editing vectors

[0119] The gRNA target sequence in the pBUE411 vector was replaced with the target sequence designed in step 1 to construct a recombinant vector, which was then sequenced. The sequencing results and the target sequence were compared and analyzed, and the recombinant vector with the correct sequence was recorded as pBUE411-KR2 / 3. The recombinant vector pBUE411-KR2 / 3 can transcribe sgRNA (SEQ ID NO.7) and Cas9 protein targeting KR2 and KR3.

[0120] 2. Transformation and identification of KR2 and KR3 CRISPR / Cas9 gene-edited plants

[0121] 1. Construction of transgenic plants

[0122] The pBUE411-KR2 / 3 recombinant vector constructed in Example 1 was introduced into the Agrobacterium EHA105 strain, and positive clones were identified by colony PCR. The correctly identified single Agrobacterium colony was inoculated into 2 mL of liquid culture medium containing 100 μg / mL kanamycin and 50 μg / mL rifampicin, and cultured overnight at 28°C with shaking. The next day, it was transferred to a large amount of liquid culture medium containing antibiotics and cultured with shaking. The cells were collected when they grew to the logarithmic phase and resuspended to OD 600 Under sterile conditions, immature embryos of maize inbred line ND101 were peeled off and infected with recombinant Agrobacterium suspension to induce maize embryo callus to seedlings. T was obtained by screening with Basta resistance medium.0 The transgenic seedlings were obtained by 0 The transgenic plants were taken as T 0 The leaves of the seedlings of the first generation of transgenic plants were tested for the copy number of the resistance gene Bar, and low-copy transgenic positive plants were screened. 0 The transgenic plants were self-pollinated to obtain T 1 The transgenic plants, T 1 The transgenic plants were self-pollinated to obtain T 2 The transgenic plants were kr2 / 3-1 and kr2 / 3-2.

[0123] 2. Identification of KR2 and KR3 CRISPR / Cas9 gene-edited plants

[0124] Extract T 2 DNA of transgenic plants and wild-type maize inbred line ND101 were collected and PCR amplified and sequenced at the target site. The PCR amplification primer sequences are as follows:

[0125] kr2-F: 5'-GGTTCTTGTTTCTTGGCGGG-3' (SEQ ID NO. 8).

[0126] kr2-R: 5'-TAAAGGTCACTGGGACAGGCTA-3' (SEQ ID NO. 9).

[0127] kr3-F: 5'-CCACACTATACCCGTGCCTC-3' (SEQ ID NO. 10).

[0128] kr3-R: 5'-TGAAAGATTCGTGATAGGTGTCAAC-3' (SEQ ID NO. 11).

[0129] The sequencing results are as follows Figure 1 As shown, the results show that: 2 T 2 The transgenic lines of the first generation were all maize mutants with homozygous mutations in the KR2 and KR3 genes (the same mutation occurred in both chromosomes).

[0130] The only difference between the kr2 / 3-1 mutant and the wild-type maize inbred line ND101 genome sequence is that two bases (d2, CA) are deleted at positions 197 and 198 of the gene encoding the KR2 protein (SEQ ID NO.2), causing a frameshift mutation in the KR2 protein; and one base (d1, T) is deleted at position 115 of the gene encoding the KR3 protein (SEQ ID NO.5), causing a frameshift mutation in the KR3 protein.

[0131] The only difference between the kr2 / 3-2 mutant and the wild-type maize inbred line ND101 genome sequence is that two bases (d2, TG) are deleted at positions 199 and 200 of the gene encoding the KR2 protein (SEQ ID NO.2), causing a frameshift mutation in the KR2 protein; and one base (d1, A) is deleted at position 114 of the gene encoding the KR3 protein (SEQ ID NO.5), causing a frameshift mutation in the KR3 protein.

[0132] T 2 The mutant strains were self-pollinated to obtain T 3 The seeds of the mutant lines were used for subsequent experiments.

[0133] Example 2: Solution culture phenotype experiment of kr2 / 3 mutant

[0134] Test materials: wild-type maize inbred line ND101 (WT), and mutant lines kr2 / 3-1 and kr2 / 3-2 prepared in Example 1.

[0135] Experimental method: The seeds of the test materials were germinated in moist vermiculite until they reached the one-leaf and one-heart stage (about 5 days). The seedlings with uniform growth were selected, the endosperm was carefully removed, and the roots of the seedlings were cleaned with deionized water. The seedlings were transplanted to HK nutrient solution for 2 days, and then HK (1.85 mM K + ) and LK (0.03 mM K + ) treatment, during which the hydroponic solution was replaced every 2 days, and the culture was ventilated. Photos and samples were taken on the 14-16th day of culture. The whole plant size and the difference in fully expanded leaves between the wild type and the mutant were compared. Hydroponic conditions: the light source was a high-intensity sodium lamp; the photoperiod was 14 hours of light / 10 hours of darkness; the light intensity was about 400 μmol / m 2 s 2 ; Temperature is 28°C in light and 23°C in darkness; Humidity is 60%.

[0136] The potassium content in normal plants is significantly reduced after low-potassium treatment, and the old leaves show yellowing edges and reduced green retention. Therefore, leaf green retention can be used as an indicator to reflect the changes in potassium content in plants after low-potassium treatment.

[0137] The results of the hydroponic experiment were as follows: Figure 3 As shown, low potassium hydroponic conditions ( Figure 3 Under the condition of LK in A, it was observed that the whole plant of the kr2 / 3 mutant material grew more robustly ( Figure 3 The second and third leaves of the control material ND101 had better green retention than kr2 / 3-1 and kr2 / 3-2, and there was no significant difference in the green retention of the fourth leaf; under high potassium hydroponic conditions ( Figure 3Under the condition of HK in B), the growth state of the whole plant of the kr2 / 3 mutant material was not significantly different from that of the control, and there was no significant difference in the leaf color from the second to the fourth leaf. This indicates that kr2 / 3 is involved in regulating the potassium ion balance of leaves (related literature on the relationship between greenness and potassium content: Xu et al., 2006, cell. A protein kinase, interacting with two calcineurin B-like proteins, regulates K+ transporter AKT1 in Arabidopsis).

[0138] Example 3, potassium content detection of kr2 / 3 mutant

[0139] 1. Biomass (dry weight) determination

[0140] The obtained corn material was dried in an oven at 80°C (3-5 days) to constant weight, and the dried material was weighed using an analytical balance to record the dry weight.

[0141] 2. Determination of potassium content

[0142] If the dry weight of the material is less than 0.2g, the sample is directly cut into pieces and placed in a crucible; if the dry weight of the material is greater than 0.2g, it needs to be crushed. The present invention uses a western medicine powder grinder to fully crush the material. After crushing, weigh about 0.2g of the sample again, and record the dry weight as w (g).

[0143] (1) Acid soaking and drying of crucible

[0144] Use a sufficient 30mL crucible and lid to prepare 0.1N HCl (measure 8.68mL of concentrated hydrochloric acid and add it to the ddH 2 HO in a 1L volumetric flask with ddHO. 2 O to 1L) and immersed overnight, then dried naturally or dried at 80℃.

[0145] (2) Carbonization and ashification of materials

[0146] Put the weighed material into a dry crucible and place it in a muffle furnace for carbonization at 300℃ for 8h and ashing at 575℃ for 10h. Close the muffle furnace and take it out carefully when the temperature drops to room temperature.

[0147] (3) Hydrochloric acid dissolution and filtration

[0148] Add 20 mL of 0.1N HCl to each crucible and dissolve overnight. During this time, use a pipette to blow and mix the solution. The next day, filter with a 0.22 μm water filter to obtain 5-10 mL of solution.

[0149] (4) Solution dilution and measurement on the machine

[0150] Use 200mM KCl and 0.1N HCl to prepare solutions with potassium concentrations of 0, 25, 50, 75, 100, 125, 150, 175 and 200μM, respectively, and put them on a microwave plasma atomic emission spectrometer to make a standard curve. Take an appropriate amount of solution for pre-determination. If it exceeds the range, dilute it and record the dilution multiple as n. Generally, dilution to above 100μM and below the range is the best. The potassium concentration determination is recorded as x (μM). Potassium content calculation formula: [K + ](mmol / g)=nx×20 / 1000 / 1000 / w=0.00002nx / w.

[0151] The biomass determination results were as follows Figure 4 As shown in A, under low potassium conditions, the dry weight of the third fully expanded leaf of the kr2 / 3 mutant material was significantly higher than that of the control material ND101; while under high potassium conditions, there was no significant difference in the dry weight of the fully expanded leaves between the kr2 / 3 mutant material and the control material.

[0152] The results of potassium content determination are as follows Figure 4 As shown in B, under low potassium hydroponic conditions, the potassium content of the third leaf of the kr2 / 3 mutant material was significantly higher than that of the control material ND101; while under high potassium hydroponic conditions, the difference in the fully expanded leaves of the kr2 / 3 mutant material was not obvious. The above results indicate that KR2 and KR3 are involved in regulating the potassium ion balance of leaves.

[0153] In summary, the KR2 and KR3 genes in corn are involved in regulating the potassium ion content of corn leaves, regulating the potassium ion distribution and balance of corn leaves. In actual production, regulating the expression of KR2 and KR3 genes can be applied to corn potassium efficient breeding and improvement.

[0154] The contents not described in detail in this specification belong to the prior art known to professional and technical personnel in this field.

[0155] Finally, it should be noted that the above description is only a preferred example of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of KR2 and KR3 proteins or their encoding genes or biological materials containing their encoding genes in any of the following aspects: P1) Application in increasing potassium ion content in plant leaves; P2) Application in regulating potassium ion balance in plants; P3) Application in increasing plant biomass; P4) Application in the breeding of plants with high potassium utilization efficiency; The amino acid sequence of the KR2 protein is any one of the following: A1) the sequence shown in SEQ ID NO.1; A2) a protein sequence having the same biological function obtained by replacing and / or deleting and / or adding one or more amino acid residues in the sequence shown in SEQ ID NO.1; A3) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1) or A2); The amino acid sequence of the KR3 protein is any one of the following: A4) the sequence shown in SEQ ID NO.4; A5) a protein sequence having the same biological function obtained by replacing and / or deleting and / or adding one or more amino acid residues in the sequence shown in SEQ ID NO.5; A6) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A4) or A5).

2. The use according to claim 1, characterized in that: The nucleotide sequence of the gene encoding the KR2 protein is any one of the following: B1) the nucleotide sequence shown in SEQ ID NO.2; B2) the nucleotide sequence shown in SEQ ID NO.3; B3) a nucleotide sequence in which one or more nucleotides are replaced, deleted and / or added as the nucleotide sequence shown in SEQ ID NO.2 and the nucleotide sequence expresses a protein with the same function; The nucleotide sequence of the gene encoding the KR3 protein is any one of the following: B4) the nucleotide sequence shown in SEQ ID NO.2; B5) the nucleotide sequence shown in SEQ ID NO.3; B6) A nucleotide sequence in which one or more nucleotides are replaced, deleted and / or added to the nucleotide sequence shown in SEQ ID NO. 2 and the nucleotide sequence expresses a protein with the same function.

3. The use according to claim 1, characterized in that: The biological material is any one of the following C1) to C9): C1) a nucleic acid molecule encoding the KR2 protein and the KR3 protein according to claim 1; C2) an expression cassette containing the nucleic acid molecule described in C1); C3) a recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2); C4) a recombinant microorganism containing the nucleic acid molecule described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3); C5) a transgenic plant cell line containing the nucleic acid molecule described in C1), or a transgenic plant cell line containing the expression cassette described in C2), or a transgenic plant cell line containing the recombinant vector described in C3); C6) transgenic plant tissue containing the nucleic acid molecule described in C1), or transgenic plant tissue containing the expression cassette described in C2), or transgenic plant tissue containing the recombinant vector described in C3); C7) a transgenic plant organ containing the nucleic acid molecule described in C1), or a transgenic plant organ containing the expression cassette described in C2), or a transgenic plant organ containing the recombinant vector described in C3); C8) a nucleic acid molecule that inhibits or reduces the expression of a gene encoding the protein of claim 1 or a nucleic acid molecule that inhibits or reduces the activity of the protein of claim 1; C9) An expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing the nucleic acid molecule described in C8).

4. The use according to claim 1, characterized in that: This is achieved by inhibiting or reducing the expression level and / or activity of KR2 protein and KR3 protein.

5. A method for improving plant potassium utilization efficiency, characterized in that: The expression amount and / or activity of KR2 protein and KR3 protein are inhibited or reduced by transgenic technology to obtain mutant plants, and the potassium utilization efficiency of plants is improved; The amino acid sequence of the KR2 protein is any one of the following: A1) the sequence shown in SEQ ID NO.1; A2) a protein sequence having the same biological function obtained by replacing and / or deleting and / or adding one or more amino acid residues in the sequence shown in SEQ ID NO.1; A3) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1) or A2); The amino acid sequence of the KR3 protein is any one of the following: A4) the sequence shown in SEQ ID NO.4; A5) a protein sequence having the same biological function obtained by replacing and / or deleting and / or adding one or more amino acid residues in the sequence shown in SEQ ID NO.4; A6) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A4) or A5).

6. A method for increasing plant biomass, characterized in that: The expression amount and / or activity of KR2 protein and KR3 protein are inhibited or reduced by transgenic technology to obtain mutant plants, and the plant biomass is increased; The amino acid sequence of the KR2 protein is any one of the following: A1) the sequence shown in SEQ ID NO.1; A2) a protein sequence having the same biological function obtained by replacing and / or deleting and / or adding one or more amino acid residues in the sequence shown in SEQ ID NO.1; A3) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1) or A2); The amino acid sequence of the KR3 protein is any one of the following: A4) the sequence shown in SEQ ID NO.4; A5) a protein sequence having the same biological function obtained by replacing and / or deleting and / or adding one or more amino acid residues in the sequence shown in SEQ ID NO.4; A6) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A4) or A5).

7. The method according to claim 5 or 6, characterized in that: The coding genes of KR2 protein and KR3 protein in corn are edited by CRISPR / Cas9 technology to inhibit the expression level and / or activity of KR2 protein and KR3 protein in corn; the gRNA target sequence is shown in SEQ ID NO.

7.

8. A KR2 and KR3 gene mutant material, characterized in that: The mutant material is any one of the following: 1) The nucleotide sequence of the gene of the kr2 / 3 mutant material is: The KR2 gene sequence shown in SEQ ID NO.2 lacks two bases CA at positions 197 and 198; The KR3 gene sequence shown in SEQ ID NO.5 has a missing base T at position 115; or 2) the nucleotide sequence of the gene of the kr2 / 3 mutant material is: The KR2 gene sequence shown in SEQ ID NO.2 lacks two bases TG at positions 199 and 200; The 114th position of the KR3 gene sequence shown in SEQ ID NO.5 is missing a base A.

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