Application of cassava potassium ion transport protein and related biological materials thereof

Abstract

The invention discloses application of cassava potassium ion transport protein and related biological materials thereof, and belongs to the technical field of biology. The technical problem to be solved by the invention is to provide a gene capable of promoting flowering of plants, enhancing salt resistance and barren resistance of the plants and increasing crop yield. The cassava potassium ion transport protein is named as MeHAK5, experiments prove that overexpression of the MeHAK5 not only improves the low potassium resistance and salt stress resistance of transgenic arabidopsis thaliana, but also shortens the flowering time of the transgenic arabidopsis thaliana and rice, and the transgenic arabidopsis thaliana and the rice head and bloom 4 days and 7 days earlier than those of non-transgenic plants respectively. Meanwhile, the effective tiller number and yield of the transgenic rice are obviously higher than those of wild rice. Therefore, the results provide basis and gene materials for cultivating barren-resistant and high-salt-resistant crop varieties, shortening the breeding cycle and improving the crop yield through genetic engineering, and have great application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to application of cassava potassium ion transport protein and related biological materials thereof. Background Art

[0002] Potassium is an essential element for plant growth and development, and is also the most abundant inorganic cation in plants. When the potassium content in the soil is too low, plants will experience serious growth defects. In addition, potassium ions are also involved in the plant's response to biotic and abiotic stresses. When plants grow in saline soils, high concentrations of Na + K absorption by plants + Competition not only affects the plant's absorption of K + , and also through outwardly rectifying potassium ion channels, leading to K + Leakage, reduce K + / Na + ratio, causing metabolic disorders in plants. Therefore, increasing K + absorption, maintaining intracellular K + Homeostasis is not only a basic requirement for plant growth and development, but also one of the important strategies for plants to resist salt stress. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a gene for promoting plant flowering, increasing the number of plant tillers, improving plant yield, and improving plant salt resistance and / or barrenness resistance. The technical problem to be solved is not limited to the described technical subject matter, and those skilled in the art can clearly understand other technical subjects not mentioned herein through the following description.

[0004] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0005] Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity and / or content of the protein in any one of D) or F),

[0006] The D) is any of the following:

[0007] D1) shorten the flowering time of plants;

[0008] D2) preparing products of plants with shortened flowering time;

[0009] D3) Cultivating plants with shortened flowering time;

[0010] D4) preparing products for breeding plants with shortened flowering time;

[0011] D5) Plant breeding;

[0012] D6) Increase the plant's tolerance to low potassium;

[0013] D7) preparing products that improve plant tolerance to low potassium;

[0014] D8) Breeding plants with improved tolerance to low potassium;

[0015] D9) preparing products for breeding plants with improved ability to tolerate low potassium;

[0016] D10) Improvement of plants with high tolerance to low potassium;

[0017] D11) Increase the ability of plants to resist salt stress;

[0018] D12) preparing products for improving the ability of plants to resist salt stress;

[0019] D13) cultivating plants with improved resistance to salt stress;

[0020] D14) preparing products for breeding plants with improved resistance to salt stress;

[0021] D15) Improving plants with high resistance to salt stress;

[0022] The F) is any of the following:

[0023] F1) Increase the number of effective tillers of plants;

[0024] F2) preparing a product for increasing the number of effective tillers of a plant;

[0025] F3) cultivating plants with increased number of effective tillers;

[0026] F4) preparing products for growing plants with an increased number of effective tillers;

[0027] F5) Increase plant yield;

[0028] F6) preparing products for increasing plant yield;

[0029] F7) Breeding plants with increased yield;

[0030] F8) preparing products for breeding plants with increased yield;

[0031] F9) Plant breeding;

[0032] The protein is cassava potassium ion protein.

[0033] The yield in F5)-F8) refers to the yield per plant.

[0034] The purpose of plant breeding mentioned above includes cultivating plants that bloom early, cultivating plants with improved low potassium tolerance, cultivating plants with improved salt stress resistance, cultivating plants with increased tillering number, and cultivating plants with increased yield. The improved plants with high low potassium tolerance and improved plants with high salt stress resistance refer to using MeHAK5 to improve the low potassium tolerance and salt stress resistance of plants.

[0035] The substances mentioned above are any of the following:

[0036] B1), a nucleic acid molecule encoding the aforementioned protein;

[0037] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0038] B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0039] B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0040] B5), a transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2);

[0041] B6), transgenic plant tissue containing the nucleic acid molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2).

[0042] The cassava potassium ion protein described above is any one of the following,

[0043] A1) a protein whose amino acid sequence is SEQ ID No. 1;

[0044] A2) a protein having more than 80% identity with the protein shown in A1) and having the same function as the protein shown in A1) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in SEQ ID No.1;

[0045] A3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

[0046] The nucleic acid molecule in B1) mentioned above is a cDNA molecule or a DNA molecule whose coding sequence is SEQ ID No.2.

[0047] In order to facilitate purification or detection of the protein in A1), a tag protein may be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID No. 1 in the sequence listing.

[0048] The tag protein includes but is not limited to: GST (glutathione sulfhydryltransferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0049] A person skilled in the art can easily mutate the nucleotide sequence encoding the protein MeHAK5 of the present invention by using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides having 75% or more identity with the nucleotide sequence of the protein MeHAK5 isolated by the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the protein MeHAK5 and have the function of the protein MeHAK5.

[0050] The aforementioned 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0051] Herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and searching to calculate the identity of an amino acid sequence, the value (%) of identity can then be obtained.

[0052] Herein, the 80% or greater identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0053] The present invention also provides a method for making a plant bloom early, comprising up-regulating or enhancing or increasing the expression level of the gene encoding the above protein or the content of the protein in the target plant to make the target plant bloom early.

[0054] The present invention also provides a method for increasing the tillering number of a plant, comprising up-regulating, enhancing or increasing the expression amount of the coding gene of the above-mentioned protein in the target plant or the content of the protein to increase the tillering number of the target plant.

[0055] The present invention also provides a method for increasing plant yield, comprising up-regulating, enhancing or increasing the expression level of the coding gene of the above-mentioned protein or the content of the protein in the target plant to increase the yield of the target plant.

[0056] Furthermore, the above-mentioned up-regulating, enhancing or increasing the expression level of the gene encoding the aforementioned protein or the content of the aforementioned protein in the target plant is to introduce the gene encoding the aforementioned protein into the target plant.

[0057] Specifically, the present invention constructs the coding gene of protein MeHAK5 (SEQ ID No. 2) into the nucleotide sequence between the Sal I and BamHI enzyme recognition sites of the pCAMBIA1300 vector and keeps other sequences unchanged to obtain a recombinant expression vector, and then transforms a receptor plant with the recombinant expression vector to obtain a transgenic plant. The transgenic plant has an earlier flowering time than the receptor plant, more tillers than the receptor plant, and a higher yield than the receptor plant.

[0058] The plant described herein is any of the following:

[0059] G1) monocots or dicots;

[0060] G2) Plants of the order Poaceae or Cruciferae;

[0061] G3) Gramineae or Cruciferae plants;

[0062] G4) a plant of the genus Oryza or the genus Arabidopsis;

[0063] G5) rice or Arabidopsis thaliana.

[0064] Also provided herein is a biomaterial, which is any of the following:

[0065] B1), a nucleic acid molecule encoding the aforementioned protein;

[0066] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0067] B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0068] B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0069] B5), a nucleic acid molecule that inhibits, reduces or down-regulates the expression of the gene encoding the protein or a nucleic acid molecule that inhibits, reduces or down-regulates the activity or content of the protein;

[0070] B6), expressing the coding gene of the nucleic acid molecule described in B5);

[0071] B7), an expression cassette containing the gene described in B6);

[0072] B8), a recombinant vector containing the gene described in B6), or a recombinant vector containing the expression cassette described in B7);

[0073] B9), a recombinant microorganism containing the gene described in B6), or a recombinant microorganism containing the expression cassette described in B7), or a recombinant microorganism containing the recombinant vector described in B4).

[0074] In the above-mentioned biological material, the expression cassette containing the nucleic acid molecule encoding MeHAK5 (MeHAK5 gene expression cassette) refers to a DNA capable of expressing MeHAK5 in a host cell, and the DNA may include not only a promoter for initiating the transcription of MeHAK5, but also a terminator for terminating the transcription of MeHAK5. Further, the expression cassette may also include an enhancer sequence. 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. Suitable transcription terminators include, but are not limited to, Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminators.

[0075] The recombinant vector containing the MeHAK5 gene expression cassette can be constructed using an existing 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, pCAMBIA1305, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Company), etc. The plant expression vector may also include the 3′ end non-translated region of the foreign gene, i.e., a polyadenylic acid signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide polyadenylic acid to be added to the 3′ end of the mRNA precursor, such as the Agrobacterium crown gall induction (Ti) plasmid gene (such as the nopaline synthase gene Nos), the non-translated region transcribed at the 3′ end of the plant gene (such as the soybean storage protein gene), all of which have similar functions. When using the gene construction plant expression vector of the present invention, 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 identical to the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The source of the translation control signal and the start codon is extensive and can be natural or synthetic. The translation initiation region can come from a transcription initiation region or a 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.

[0076] In the above biological materials, the vector is well known to those skilled in the art, including but not limited to: plasmid, phage (such as lambda phage or M13 filamentous phage, etc.), cosmid (i.e. cosmid), Ti plasmid or viral vector. Specifically, it can be p416-GPD or pCAMBIA1300.

[0077] In the above biological materials, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0078] The nucleic acid molecule described in B1) above is a cDNA molecule or a DNA molecule whose coding sequence is SEQ ID No.2.

[0079] The proteins mentioned above also fall within the protection scope of the present invention.

[0080] HAK is a class of high-affinity potassium ion transporter proteins distributed on plant cell membranes. + Through bioinformatics analysis, a gene encoding a high-affinity K+ transport protein was screened from cassava, named MeHAK5. Experiments have shown that the expression of this gene improves the potassium absorption capacity of the potassium absorption-deficient yeast strain CY162 ( Figure 1 ), the corresponding expression of MeHAK5 not only improves the tolerance of transgenic Arabidopsis to low potassium ( Figure 2 ) and salt stress resistance ( Figure 3 ) and shortened the transgenic Arabidopsis ( Figure 4 ) and rice ( Figure 5 and Figure 6 ) flowering time, transgenic Arabidopsis and rice heading and flowering 4 and 7 days earlier than non-transgenic plants, respectively. In addition, the number of tillers and yield of transgenic rice were significantly increased compared with wild-type rice. Therefore, these results provide a basis and genetic materials for breeding crop varieties that are tolerant to barrenness and high salt resistance, shorten the breeding cycle, increase the number of tillers and improve yield through genetic engineering, and have great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 The MeHAK5 gene complemented the sensitivity of the CY162 yeast strain to low potassium stress.

[0082] Figure 2 Overexpression of MeHAK5 promotes the growth of transgenic Arabidopsis under low potassium conditions. WT, wild-type Arabidopsis; HAK5, Arabidopsis transgenic with MeHAK5 gene.

[0083] Figure 3 Overexpression of MeHAK5 promotes the growth of transgenic Arabidopsis under salt stress. WT, wild-type Arabidopsis; HAK5, Arabidopsis transgenic with MeHAK5 gene.

[0084] Figure 4 MeHAK5 promotes early flowering in transgenic Arabidopsis.

[0085] Figure 5 MeHAK5 promotes early flowering in laboratory-grown transgenic rice.

[0086] Figure 6 MeHAK5 promoted early flowering of field-grown transgenic rice (MeHAK5OE-1 and MeHAK5OE-2).

[0087] Figure 7 This is the maturity phenotype of MeHAK5 transgenic rice and wild-type rice planted in the field in Lingshui.

[0088] Figure 8 This is the appearance of rice grown under laboratory conditions.

[0089] Fig. 9 These are grains of rice grown under laboratory conditions.

[0090] Fig.10 Statistical analysis of data for MeHAK5 transgenic rice grown in the laboratory.

[0091] Fig.11 MeHAK5 promoted early flowering of field-grown transgenic rice (HAK5-1, HAK5-2, and HAK5-3).

[0092] Fig.12 Rice grown in the fields. DETAILED DESCRIPTION

[0093] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0094] 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.

[0095] pMD-18T vector, p416-GPD, pCAMBIA1300-GFP vector: described in the non-patent literature “Xiuzhen Chen, Minghua Luo, Chunyan Mo, Wenjia Li, Yiying Ji, Qing Xie, Xingyu Jiang. MeCIPK10 regulates the transition of the K +transport activity of MeAKT2 between low- and high-affinity molds in cassava. Journal of Plant Physiology, 2023, 280: 153861”, which is available to the public from Guangdong Ocean University. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0096] Example 1. Cloning of MeHAK5

[0097] The materials used include: PrimeSTAR Max DNA Polymerase (TaKaRa); Recombination Cloning Kit (Vazyme); DH5a competent cells (Biomed); Ampicillin (Sangon); Kanamycin (Kanamycin); T4 DNA ligase (NEB)

[0098] Total RNA of cassava (Huanan No. 8) was extracted and reverse transcribed into cDNA. The MeHAK5 gene was amplified by PCR (polymerase chain reaction) using cDNA as a template and MeHAK5-F as an upstream primer and MeHAK5-R as a downstream primer. The gene fragment was connected to the pMD-18T vector by TA cloning, and the obtained recombinant plasmid was named p18-MeHAK5. Finally, the plasmid p18-MeHAK5 was transformed into Escherichia coli for amplification and preservation.

[0099] MeHAK5-F: 5′-GAAGAAGATGGCAGAGGAAGTG-3′;

[0100] MeHAK5-R: 5′-GCAGCATCATCATCTCATATGTC-3′.

[0101] The amino acid sequence of MeHAK5 is SEQ ID No.1, and the nucleotide sequence is SEQ ID No.2.

[0102] Example 2, K of MeHAK5 + Transshipment Capacity

[0103] 1. Construction of yeast expression vector

[0104] Using the plasmid p18-MeHAK5 constructed in Example 1 as a template, first use the upstream primer p416-F and the downstream primer p416-R to perform a PCR reaction. PCR reaction procedure: the first step is 98°C for 3 minutes; the second step is 34 cycles, the cycle is 98°C for 10 seconds, 58°C for 15 seconds, and 72°C for 2.5 minutes; the third step is 72°C for 10 minutes. Purify the PCR product, then use Sal I and BamHI to digest the p416-GPD vector and the target fragment, recover the target fragment and connect it using T4 ligase, use the heat shock method to transform the ligation product into DH5a competent cells, and use ampicillin antibiotics to screen transgenic Escherichia coli DH5a carrying the recombinant plasmid p416-MeHAK5. The transformation product clones are sent to a bioengineering sequencing company for DNA sequencing analysis to determine the correct transformants. Extract plasmid p416-MeHAK5 from transgenic Escherichia coli DH5a containing the recombinant plasmid p416-MeHAK5.

[0105] 2. MeHAK5 gene complementation of CY162 yeast strain's sensitivity to low potassium stress

[0106] CY162 (trk1Δ, trk2Δ) is a yeast mutant strain deficient in potassium absorption. + It cannot grow when the concentration is lower than 5mM, so it is used for K + A good tool for heterologous verification of absorption function. The potassium ion-deficient yeast strain CY162 was transformed using the LiAc / PEG method. The specific method is as follows:

[0107] p416-MeHAK5 recombinant plasmid ( Figure 1 , two replicates) were transformed into yeast mutant strain CY162 as the experimental group, and yeast CY162 transformed with empty vector p416-GPD was used as the control ( Figure 1 ). The results of the experiment were performed on AP solid medium (10 mM arginine, 8 mM phosphate, 2% glucose, 2 mM MgS04, 0.2 mM CaCl2, trace minerals and vitamins, 2% agar) containing different potassium ion concentrations. + Growth of transgenic yeast under different concentration conditions.

[0108] The results are as follows Figure 1 As shown, when K + All transgenic yeasts grew well and had no difference when the concentration was ≥5mM, but when K + When the concentration dropped to 1 mM, the differences between the different transgenic yeasts were significant. The control yeast could not grow, but the MeHAK5-transfected yeast still grew well. +When the concentration was reduced to 0.1 mM, the transgenic yeast expressing MeHAK5 showed a weakened growth but still grew well. The above experimental results show that MeHAK5 is a high-affinity potassium ion transporter that can help transgenic yeast absorb potassium ions and promote the growth of transgenic yeast under low potassium conditions.

[0109] 3. Construction of plant expression vector p1300-MeHAK5

[0110] Using the p18-MeHAK5 constructed in Example 1 as a template, first use the upstream primer p1300-F and the downstream primer p1300-R to perform a PCR reaction. PCR reaction procedure: the first step is 95°C for 3 minutes; the second step is 34 cycles, the cycle is 95°C for 10 seconds, 58°C for 15 seconds, and 72°C for 2.5 minutes; the third step is 72°C for 10 minutes. Purify the PCR product, then use Sal I and BamHI to digest the pCAMBIA1300-GFP vector and the target fragment, recover the target fragment and connect it using T4 ligase, then use the heat shock method to transform the ligation product into DH5a competent cells, and use kanamycin to screen the transgenic Escherichia coli DH5a carrying the recombinant plasmid p1300-MeHAK5. The transformation product clones are sent to a bioengineering sequencing company for DNA sequencing analysis to determine the correct transformants. The recombinant plant expression vector in the correct transformant was named p1300-MeHAK5, which is a recombinant expression vector in which the nucleotide sequence between the Sal I and BamHI restriction sites of the pCAMBIA1300 vector is replaced by a DNA molecule having a nucleotide sequence of SEQ ID NO.2 and other sequences are kept unchanged, and the recombinant expression vector expresses the protein MeHAK5 having an amino acid sequence of SEQ ID No.1. The expression of the MeHAK5 gene is driven by the CaMV35S promoter.

[0111] Table 1 Primers used in two PCRs

[0112] Upstream primer p416-F 5′-CGGGATCCGAAGAAGATGGCAGAGGAAGTG-3′ Downstream primer p416-R 5′-TCCCCCGGGGCAGCATCATATCTCATATGTC-3′ Upstream primer p1300-F 5′-AGCGTCGACGAAGAAGATGGCAGAGGAAGTG-3′ Downstream primer p1300-R 5′-CGGGATCCGCAGCATCATATCTCATATGTC-3′

[0113] Example 3: Overexpression of MeHAK5 promotes the growth of transgenic Arabidopsis thaliana under low potassium and salt stress conditions

[0114] 1. Genetic transformation and screening of Arabidopsis and rice

[0115] Arabidopsis thaliana (Columbia wild type, WT): recorded in the non-patent literature "The protein kinase complex CBL10-CIPK8-SOS1 functions in Arabidopsis to regulate salt tolerance. Journal of Experimental Botany, 2020, 71: 1801-1814." The public can obtain it from Guangdong Ocean University. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0116] Rice TP309, also known as rice Taipei 309, is a rice variety originating from Taiwan Province, China, hereinafter referred to as wild rice, belonging to japonica rice: recorded in the non-patent document "Initiation of Androgenesis in Rice Oryza sativa L var Taipei 309. Proceedings of Indian National Science Academy, 2015, 53 (3B)." The public can obtain it from Guangdong Ocean University. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0117] The p1300-MeHAK5 plant expression vector constructed in Example 2 was transformed into Arabidopsis thaliana (Columbia wild type, WT) through Agrobacterium tumefaciens-mediated transformation, and the harvested transgenic plants were molecularly identified using the upstream primer p1300-F and the downstream primer p1300-R, and homozygous strains were screened. After screening and identification, Arabidopsis thaliana transgenic plants of the T3 generation were obtained.

[0118] By means of Agrobacterium tumefaciens infection, p1300-MeHAK5 constructed in Example 2 was transferred into wild-type rice callus cells, and then the transformed rice callus cells were differentiated and regenerated by tissue culture technology to obtain transgenic rice plants. The harvested transgenic plants were molecularly identified using the upstream primer p1300-F and the downstream primer p1300-R, and homozygous strains were screened. After screening and identification, the T3 generation homozygous rice transgenic plants were obtained.

[0119] 1. Detection and screening of transgenic Arabidopsis

[0120] 1) The harvested T0 transgenic Arabidopsis seeds were divided into 2 mL centrifuge tubes, washed with 75% alcohol for 30 seconds, the supernatant was removed, and washed with sterile ddH2O for 3 times.

[0121] 2) Wash with 10% sodium hypochlorite for 10 min, invert several times, remove the supernatant, and add sterile ddH2O to wash 5 times.

[0122] 3) After the sterilized seeds were vernalized at 4°C in the dark for 3 days, they were evenly spread on 1 / 2MS solid medium containing 50μg / L hygromycin. The composition of 1 / 2MS solid medium is: M519: 2.215g / L, sucrose: 15g / L, agar: 7g / L, hygromycin: 50μg / L, and the balance is deionized water. M519 was purchased from PhytoTech LABS, item number M519-50L.

[0123] 4) Place the plate with the seeds flat in a lighted plant incubator at 23°C, 16h light / 8h dark. After two weeks of incubation, select the seedlings with roots and true leaves and transplant them into the soil for further growth.

[0124] The above method was followed until homozygous Arabidopsis transgenic plants of the T3 generation were obtained.

[0125] 2. Detection and screening of genetically modified rice

[0126] The obtained T0 generation transgenic rice seedlings were planted in the greenhouse of Hainan University. After about 15 days of planting, young leaves were taken and genomic DNA was extracted for PCR detection. The genomic DNA extraction method was as follows:

[0127] 1) Take about 0.1 g of young fresh leaves, put them into a 1.5 mL centrifuge tube, and grind them into powder with liquid nitrogen.

[0128] 2) Add 700 μL of preheated 2% CTAB extraction buffer and place in a 65°C water bath for about 30 minutes, mixing by inverting every 10 minutes.

[0129] 3) After the mixture has cooled to room temperature, add 700uL of (chloroform: isoamyl alcohol = 24:1) solution and mix well. Gently invert the centrifuge tube several times to make the mixture in the tube into an emulsion. Centrifuge at 10000rpm for 10min at room temperature and transfer the supernatant to another clean centrifuge tube.

[0130] 4) Add 2.5 times the volume of pre-cooled anhydrous ethanol to the supernatant, mix well, place in a 4°C refrigerator for 30 min, centrifuge at 12000 rpm for 10 min, and discard the supernatant.

[0131] 5) Add 750uL 75% ethanol to wash the precipitate, centrifuge at 10000rpm for 5min, discard the supernatant, and wash the precipitate twice.

[0132] 6) Invert or place in a fume hood to dry.

[0133] 7) Add 100u LTE buffer to dissolve the DNA and store at -4℃ for later use.

[0134] The above DNA was used as a template, and p1300-F was used as an upstream primer and p1300-R was used as a downstream primer for PCR reaction. The PCR reaction procedure was as follows: the first step was 95°C for 3 minutes; the second step was 34 cycles, with the cycles being 95°C for 15 seconds, 58°C for 15 seconds, and 72°C for 1 minute; the third step was 72°C for 10 minutes. Detection was performed using 1% agarose gel, and the corresponding plants with the same band size were selected and continued to be cultured, and the above method was used until the T3 generation homozygous transgenic rice plants were screened.

[0135] The homozygous transgenic plants identified above were named as Arabidopsis thaliana T3 homozygous plants MeHAK5OE-11 and MeHA K5OE-15, and rice T3 homozygous plants MeHAK5OE-1 and MeHAK5OE-2.

[0136] 2. MeHAK5 overexpression promotes the growth of transgenic Arabidopsis thaliana under low potassium and salt stress conditions

[0137] 1. Experimental methods

[0138] 1 / 2MS solid culture medium: purchased from PhytoTech LABS, product number M153-50L.

[0139] Low potassium medium containing 10 μM potassium chloride (KCl): ammonium nitrate (NH4NO3) 2.5 g / L, diammonium phosphate [(NH4)2HPO4] 0.165 g / L, M407 0.61 g / L, potassium chloride 10 μM, and the remainder is deionized water. M407 was purchased from PhytoTech LABS, item number M407-50L.

[0140] The high salt medium containing 100 mM sodium chloride (NaCl) is composed of: M519 2.215 g / L, sodium chloride 100 mM, and the balance is deionized water. M519 was purchased from PhytoTech LABS, item number M519-50L.

[0141] Wild type (WT) -10μK + Group: Wild-type Arabidopsis thaliana (WT) was sown on 1 / 2MS solid medium and grown in a light incubator for 5 days. The light incubator conditions were 23°C, 16 h light / 8 h dark. Then 5 wild-type seedlings were transplanted to a low potassium medium containing 10 μM KCl (i.e. Figure 2 Medium 10μK + ), and then grown in a light incubator for two weeks without changing the culture medium. The light incubator conditions were 23°C, 16 h light / 8 h dark, and the growth and phenotype of Arabidopsis were observed. The experiment was repeated three times.

[0142] Wild type (WT)-1 / 2MS group: 1 / 2MS solid medium was used instead of wild type (WT)-10μK + The low potassium medium in the group contained 10 μM KCl, and the rest of the operations were the same as those in the wild type (WT)-10 μM KCl. + Group.

[0143] Arabidopsis transgenic plants (MeHAK5OE-11 and MeHAK5OE-15) -10μK + Group: Arabidopsis thaliana transgenic plants T3 homozygous MeHAK5OE-11 (4 plants) and MeHAK5OE-15 (4 plants) replaced wild type (WT) -10μK + The Arabidopsis wild type in the group, and the other conditions were the same as the wild type (WT) -10μK + Group.

[0144] Arabidopsis transgenic plants (MeHAK5OE-11 and MeHAK5OE-15)-1 / 2MS group: 1 / 2MS solid medium was used instead of Arabidopsis transgenic plants (MeHAK5OE-11 and MeHAK5OE-15)-10μK + The low potassium medium in the group contained 10 μM KCl, and the rest of the operations were the same as those of the wild type WT-10 μK + Group.

[0145] Wild-type (WT)-100 mM NaCl group: Wild-type Arabidopsis thaliana (WT) was sown on 1 / 2MS solid medium and grown in a light incubator for 5 days. The light incubator conditions were 23°C, 16 h light / 8 h dark. Then, 5 wild-type seedlings were transplanted to a high-salt medium containing 100 mM NaCl (i.e. Figure 3 The Arabidopsis thaliana was grown in a light incubator for two weeks without changing the medium. The conditions of the light incubator were 23°C, 16 h light / 8 h dark, and the phenotype was observed. The experiment was repeated three times.

[0146] Wild-type (WT)-1 / 2MS group: The high-salt medium containing 100 mM NaCl in the wild-type (WT)-100 mM NaCl group was replaced with 1 / 2MS solid medium. The rest of the operations were the same as those in the wild-type-10 μK + Group.

[0147] Arabidopsis transgenic plants (MeHAK5OE-11 and MeHAK5OE-15)-100 mM NaCl group: Arabidopsis wild type in the wild type (WT)-100 mM NaCl group was replaced by Arabidopsis transgenic plants T3 homozygous MeHAK5OE-11 (5 plants) and MeHAK5OE-15 (5 plants), and the other conditions were the same as those of the wild type (WT)-10 μK + Group.

[0148] Arabidopsis transgenic plants (MeHAK5OE-11 and MeHAK5OE-15)-1 / 2MS group: The high-salt medium containing 100 mM NaCl in the Arabidopsis transgenic plants (MeHAK5OE-11 and MeHAK5OE-15)-100 mM NaCl group was replaced with 1 / 2MS solid medium, and the rest of the operations were the same as the wild type-100 mM NaCl group.

[0149] 2. Experimental results: The results are as follows Figure 2 and Figure 3 As shown, there was no significant difference in the phenotypes of the two on the control 1 / 2MS solid medium, but there was a significant phenotypic difference between the two on low potassium and high salt medium ( Figure 2 and Figure 3 ), the transgenic Arabidopsis thaliana grew better than the wild type and had a more developed root system, indicating that MeHAK5 can promote the growth of transgenic Arabidopsis thaliana under low potassium or salt stress conditions.

[0150] Example 4: Overexpression of MeHAK5 promotes early flowering of transgenic Arabidopsis and rice

[0151] 1. MeHAK5 promotes flowering in transgenic Arabidopsis

[0152] The wild-type Arabidopsis thaliana (WT) and transgenic Arabidopsis thaliana expressing MeHAK5 (MeHAK5OE-1, MeHAK5OE-2) obtained in Example 3 were sown in pots filled with nutrient soil (purchased from PINDSTRUP, normal soil ion content), with 3 replicates per group, and grown in a 22°C greenhouse with 16h light / 8h dark, and the rest of the operations were the same. The heading and flowering time of Arabidopsis thaliana was observed and photographed and recorded ( Figure 4 ), the results showed that the transgenic Arabidopsis thaliana began to head and flower 28 days after sowing, while the wild-type Arabidopsis thaliana began to head and flower 32 days after sowing, 4 days earlier than the wild-type plants.

[0153] MeHAK5 promotes flowering of transgenic laboratory-grown rice

[0154] The wild-type rice obtained in Example 3 and the T3 homozygous transgenic rice expressing MeHAK5 (MeHAK5OE-1 and MeHAK5OE-2) were sown in pots filled with nutrient soil (purchased from PINDSTRUP, normal soil ion content), with 3 replicates per group and 4 seedlings per replicate. They were sown on July 23, 2022 and grown naturally at the experimental base of Hainan University in Haikou. The rest of the operations were the same. Observe the heading and flowering time of rice and take photos to record ( Figure 5 ), from left to right in the figure are MeHAK5OE-1, MeHAK5OE-2 and WT. The results showed that the transgenic rice began to head and flower 72 days after sowing, while the wild-type rice began to head and flower 78 days after sowing. The transgenic rice flowered 7 days earlier than the wild-type plants.

[0155] 3. MeHAK5 expression transgenic rice promotes early flowering in field-grown rice

[0156] The wild-type rice obtained in Example 3 and the transgenic rice expressing MeHAK5 (MeHAK5OE-1 and MeHAK5OE-1) of the T3 generation were sown at the Lingshui Nanfan breeding base in Hainan on November 16, 2022 (normal soil ion content). Each type of rice to be tested was planted in three replicate plots, with 20 m2 per plot. 2 , randomly arranged and grown naturally in the field. Figure 6 (Rice grown in the field was moved to pots for comparison and photographed) As shown in the figure, from left to right are WT, MeHAK5OE-1 and MeHAK5OE-2. The flowering time of MeHAK5 transgenic Arabidopsis and rice is significantly earlier than that of the wild type. The maturity phenotypes of WT and MeHAK5 transgenic rice planted in the field in Lingshui are shown in Figure 7 As shown, the maturity time of wild-type rice planted at the same time was significantly later than that of transgenic rice expressing MeHAK5 (MeHAK5OE-1 and MeHAK5OE-1).

[0157] Example 5: Overexpression of MeHAK5 significantly increases the yield of transgenic rice

[0158] 1. Preparation of transgenic rice

[0159] Rice transgenic plants were obtained by referring to the method described in Example 3. The homozygous transgenic plants identified above were named as rice transgenic plant T3 homozygous HAK5-1, HAK5-2, HAK5-3, OEMeHAK5-1, OEMeHAK5-2 and OEMeHAK 5-3.

[0160] 2. Increased Yield of MeHAK5-Expressing Transgenic Rice Grown in the Laboratory

[0161] The experiment was repeated three times, and each repetition was as follows:

[0162] HAK5-1 group: After the seeds of the transgenic rice T3 generation homozygous line HAK5-2 were raised, they were transplanted into pots (4 plants per pot), a total of 15 pots. Then they were placed in an open-air pool and grown under natural conditions. Irrigation, fertilization, and disease prevention and control were implemented in accordance with normal field management of rice cultivation. At harvest, the number of effective tillers per plant, plant height, biomass per plant, ear length, and fruiting rate were measured; after the rice was dried, agronomic traits such as single plant seed yield and thousand-grain weight were measured, and relevant indicators were statistically analyzed.

[0163] HAK5-2 group: The HAK5-1 seeds in the HAK5-1 group were replaced with the T3 homozygous HAK5-2 mentioned above, and the rest of the operations were the same as those in the HAK5-1 group.

[0164] WT group: HAK5-1 seeds in the HAK5-1 group were replaced with WT, and the rest of the operations were the same as those in the HAK5-1 group.

[0165] Effective tiller number: refers to the number of tillers that can produce heading in a rice plant.

[0166] The results showed that although the agronomic traits of transgenic rice, such as plant height, panicle length, fruit set rate, and 1000-grain weight, were better than those of wild-type rice to a certain extent, they were statistically different between transgenic rice and wild-type rice TP309 ( Figure 8 and Fig. 9 The difference between the two transgenic lines was not significant; the biomass, effective tiller number and seed yield of the transgenic rice were significantly higher than those of the wild-type rice TP309. Equivalent to the wild-type rice TP309, the biomass, effective tiller number and seed yield of the two transgenic lines increased by 36.5% and 57.4%, 56.3% and 50%, and 26.7% and 31.77%, respectively ( Figure 8-Figure 10 ).

[0167] 3. Increased Yield of MeHAK5-Expressing Transgenic Rice Planted in Field

[0168] The experiment was repeated three times, and each repetition was as follows:

[0169] OEMeHAK5-1 group: The T3 generation homozygous seed of transgenic rice MeHAK5-3 was raised and transplanted into the field, with a wide row spacing of 40 cm, a narrow row spacing of 20 cm, and a plant spacing of 20 cm. Each rice to be tested was planted in 3 replicate plots, each with a 20m 2, randomly arranged. Grown under natural conditions, irrigation, fertilization and disease control follow the normal field management of rice cultivation. At harvest, the number of effective tillers per plant, plant height, ear length and fruit setting rate were measured; after the rice was dried, the agronomic traits such as seed yield per plant and thousand-grain weight were measured, and the relevant indicators were statistically analyzed.

[0170] OEMeHAK5-2 group: The OEMeHAK5-1 seeds in the OEMeHAK5-1 group were replaced with the T3 generation homozygous OEMeHAK5-2 mentioned above, and the rest of the operations were the same as those for the OEMeHAK5-1 group.

[0171] OEMeHAK5-3 group: The OEMeHAK5-1 seeds in the OEMeHAK5-1 group were replaced with the T3 generation homozygous OEMeHAK5-3 mentioned above, and the rest of the operations were the same as those for the OEMeHAK5-1 group.

[0172] WT group: OEMeHAK5-1 seeds in the OEMeHAK5-1 group were replaced with WT, and the rest of the operations were the same as those in the OEMeHAK5-1 group.

[0173] The results showed that the agronomic traits of MeHAK5 transgenic rice, such as plant height, panicle length, fruit set rate, and 1000-grain weight, were better than those of wild-type rice TP309 ( Figure 11-Figure 12 WT in the transgenic rice), but the difference between them and wild-type rice TP309 was not significant at the statistical level; the effective tiller number and seed yield of transgenic rice were significantly higher than those of wild-type rice TP309. Compared with the wild-type rice TP309 in the control, the effective tiller number of the three transgenic lines increased by 23.91%, 43.48% and 38.04%, respectively; the seed yield per plant increased by 21.12%, 33.83% and 29.53%, respectively (Table 2 and Fig.12 ).

[0174] Table 2 Field data statistics

[0175] Plant TP309 OEMeHAK5-1 OEMeHAK5-2 OEMeHAK5-3 Plant height 101.00±9.52 94.89±10.39 96.11±6.71 97.33±5.75 Spike length 24.69±1.38 24.60±1.96 23.94±1.85 24.62±2.30 Number of grains per ear 160.22±44.08 164.44±38.20 166.00±31.85 154.33±29.86 Number of tillers 11.78±2.57 13.56±2.06 15.78±2.20** 15.00±3.37* Effective tillering 10.22±2.04 12.67±2.11* 14.67±2.40** 14.11±3.73* Thousand-grain weight 29.21±0.79 28.69±0.82 30.17±0.66* 28.90±0.63 Fruiting rate 75.01±1.83 76.90±2.22 78.01±1.94** 75.19±2.37 Yield per plant 36.67±9.53 44.41±5.26 49.08±9.63* 47.50±7.52*

[0176] Note: OEMeHAK5-1 is Figure 8-Figure 12 HAK5-1 or OEMeHAK5-1 in OEMeHAK5-2 is Figure 8-Figure 12 HAK5-2 or OEMeHAK5-2. OEMeHAK5-3 is Fig. 9 and Fig.12 HAK5-3 or OEMeHAK5-3.

[0177] SEQ ID No.1(808aa):

[0178] 。

[0179] SEQ ID No.2(2427bp):

[0180]

[0181] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Use of a protein or a substance for regulating the expression of a gene encoding the protein or a substance for regulating the activity and / or content of a protein in any of the following D) or F), The D) is any of the following: D1) shorten the flowering time of plants; D2) preparing products of plants with shortened flowering time; D3) Cultivating plants with shortened flowering time; D4) preparing products for breeding plants with shortened flowering time; D5) Plant breeding; D6) Increase the plant's tolerance to low potassium; D7) preparing products that improve plant tolerance to low potassium; D8) Breeding plants with improved tolerance to low potassium; D9) preparing products for breeding plants with improved ability to tolerate low potassium; D10) Improvement of plants with high tolerance to low potassium; D11) Increase the ability of plants to resist salt stress; D12) preparing products for improving the ability of plants to resist salt stress; D13) cultivating plants with improved resistance to salt stress; D14) preparing products for breeding plants with improved resistance to salt stress; D15) Improving plants with high resistance to salt stress; The F) is any of the following: F1) Increase the number of effective tillers of plants; F2) preparing a product for increasing the number of effective tillers of a plant; F3) cultivating plants with increased number of effective tillers; F4) preparing products for growing plants with an increased number of effective tillers; F5) Increase plant yield; F6) preparing products for increasing plant yield; F7) Breeding plants with increased yield; F8) preparing products for breeding plants with increased yield; F9) Plant breeding; The protein is cassava potassium ion transport protein.

2. The use according to claim 1, characterized in that The substances are any of the following: B1), a nucleic acid molecule encoding the protein according to claim 1; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5), a transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2); B6), transgenic plant tissue containing the nucleic acid molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2).

3. The use according to claim 1 or 2, characterized in that The cassava potassium ion protein is any one of the following, A1) a protein whose amino acid sequence is SEQ ID No. 1; A2) a protein having more than 80% identity with the protein shown in A1) and having the same function as the protein shown in A1) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in SEQ ID No.1; A3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

4. The use according to claim 2, characterized in that: B1) The nucleic acid molecule is a cDNA molecule or a DNA molecule whose coding sequence is SEQ ID No.

2.

5. A method for causing a plant to bloom early, increase the number of effective tillers or / and increase yield, comprising upregulating or enhancing or increasing the expression of a gene encoding the protein of claim 1 or the content of the protein in a target plant so that the target plant blooms early, increases the number of effective tillers or / and increases yield.

6. The method according to claim 5, characterized in that The up-regulation, enhancement or increase of the expression level of the gene encoding the protein of claim 1 or the content of the protein in the target plant is to introduce the gene encoding the protein of claim 1 into the target plant.

7. The use according to any one of claims 1 to 4, and / or the method according to claim 5 or 6, wherein the plant is any one of the following: G1) monocots or dicots; G2) Plants of the order Poaceae or Cruciferae; G3) Gramineae or Cruciferae plants; G4) a plant of the genus Oryza or the genus Arabidopsis; G5) rice or Arabidopsis thaliana.

8. Biomaterial, characterized in that The biological material is any of the following: B1), a nucleic acid molecule encoding the protein according to claim 1; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5), a nucleic acid molecule that inhibits, reduces or down-regulates the expression of the gene encoding the protein or a nucleic acid molecule that inhibits, reduces or down-regulates the activity or content of the protein; B6), expressing the coding gene of the nucleic acid molecule described in B5); B7), an expression cassette containing the gene described in B6); B8), a recombinant vector containing the gene described in B6), or a recombinant vector containing the expression cassette described in B7); B9), a recombinant microorganism containing the gene described in B6), or a recombinant microorganism containing the expression cassette described in B7), or a recombinant microorganism containing the recombinant vector described in B4).

9. The biomaterial according to claim 8, characterized in that B1) The nucleic acid molecule is a cDNA molecule or a DNA molecule whose coding sequence is SEQ ID No.

2.

10. The protein of claim 1.

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