CN117024538A - Corn lodging-resistant gene and application of related protein and biological material thereof - Google Patents
Corn lodging-resistant gene and application of related protein and biological material thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/8255—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving lignin biosynthesis
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
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Abstract
The application discloses a corn lodging-resistant gene and application of related proteins and biological materials thereof. The technical scheme of the application is that the protein is corn MDR1 which is applied to the reduction of plant lodging rate and the improvement of plant stalk bending force and lignin content, and the amino acid sequence of the protein can be SEQ ID NO. 1 in a sequence table. Experiments prove that after the corn MDR1 gene is excessively expressed in corn, the content of lignin in the corn stalk part can be increased, and the potassium content of corn leaves can be regulated and controlled, so that the stalk bending force of the corn stalk is increased, the lodging rate of a corn plant under the condition of strong wind is reduced, and the lodging resistance of the corn plant is improved. The application has great application value in crop molecular improvement, and can be used for improving crop lodging resistance and crop lodging resistance breeding and improvement.
Description
Technical Field
The application relates to the technical field of biology, in particular to a corn lodging-resistant gene and application of related proteins and biological materials thereof.
Background
Lodging of the aerial parts of the corn in the growing period obviously affects the corn yield, and the lodging-resistant character of the corn is one of important indexes of good corn varieties. The analysis of the molecular genetic mechanism of the lodging-resistant character of the corn is of great significance for cultivating new varieties of lodging-resistant corn to ensure high and stable yield of the corn.
At present, the application of plant semi-dwarfing traits and the improvement of internode mechanical strength are widely regarded as effective methods for enhancing crop lodging resistance. The composition of the plant cell wall greatly affects the mechanical strength of the plant, with higher lignin levels playing a more important role in crop lodging resistance.
Potassium is a mineral nutrient element necessary for plant growth and development. As the first large cation in the plant body, potassium ion is an activator of enzymatic reaction in the plant body, promotes protein synthesis and photosynthesis, and participates in osmotic adjustment and maintains in-vivo charge balance and membrane potential stability. In agricultural production, sufficient potassium fertilizer supply can effectively improve the lodging resistance of crops, but the molecular mechanism of regulating and controlling lodging resistance of corns by potassium nutrition is not clear.
At present, the research on the action mechanism of the protein kinase mediated phosphorylation modification related to potassium nutrients in the plant stalk strength maintenance and lodging resistance is still fresh, so that the research on the regulation mechanism of the protein kinase on the lodging resistance of plants has important theoretical significance and research value for cultivating new lodging-resistant corn varieties so as to ensure high and stable corn yield.
Disclosure of Invention
The technical problem to be solved by the application is how to improve the lodging resistance of plants and/or how to reduce the lodging rate of plants and/or how to improve the bending force of plant stalks and/or how to improve the lignin content of plant stalks and/or how to regulate the potassium ion balance of plants and/or how to regulate the potassium ion content of plant leaves and/or how to cultivate lodging-resistant plants.
In order to solve the above technical problems, the present application provides, first of all, any one of the following uses of a protein and/or a substance regulating the expression of a gene encoding the protein and/or a substance regulating the content of the protein and/or a substance regulating the activity of the protein:
p1, in regulating and controlling or reducing plant lodging rate,
p2, in regulating and controlling or improving the bending force of plant stems,
p3, in regulating and controlling or improving the lignin content of the plant stalks,
p4, in regulating and controlling or promoting the potassium ion balance of plants,
p5, in regulating and controlling or improving the potassium ion content of plant leaves,
p6, application in plant lodging-resistant breeding,
p7, application in plant quality improvement.
The protein may be a protein of the following A1), A2) or A3):
a1 Amino acid sequence is protein of sequence 1 in a sequence table;
a2 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the sequence 1 in the sequence table, is derived from A1) and has the same function or has more than 80 percent of the same with the protein shown in A1) and has the same function;
a3 Fusion proteins obtained by ligating protein tags at the N-terminus or/and the C-terminus of A1) or A2).
The proteins described above may be derived from maize.
The protein can be synthesized artificially or obtained by synthesizing the coding gene and then biologically expressing.
Among the above proteins, the protein tag (protein-tag) refers to a polypeptide or protein that is fusion expressed together with a target protein by using a DNA in vitro recombination technique, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc.
In the above proteins, the identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, the identity of a pair of amino acid sequences can be searched for by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, per residue gap cost and Lambda ratio to 11,1 and 0.85 (default values), respectively, and calculating, and then obtaining the value (%) of the identity.
In the above protein, the 80% or more identity may be at least 81%, 82%, 85%, 86%, 88%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity. The plant described above may be any of the following:
c1 Dicotyledonous plants;
d1 A monocotyledonous plant,
d2 A plant of the family Gramineae,
d3 A plant of the order Gramineae,
d4 A maize plant;
d5 Corn).
The above-mentioned substances may be the following biological materials.
The modulation may be up-regulation or enhancement or improvement.
In order to solve the above technical problems, the present application also provides any one of the following applications of the biological material related to the above protein:
q1, the application of the biological material in regulating and controlling or reducing the plant lodging rate,
q2, the application of the biological material in regulating and controlling or improving the bending force of plant stems,
q3, the application of the biological material in regulating and controlling or improving the lignin content of the plant stalks,
q4, the application of the biological material in regulating or promoting the potassium ion balance of plants,
q5, the application of the biological material in regulating and controlling or improving the potassium ion content of plant leaves,
q6, the application of the biological material in plant lodging-resistant breeding,
q7, the application of the biological material in plant quality improvement.
The biomaterial may be any one of the following B1) to B9):
b1 A nucleic acid molecule encoding a protein as described above;
b2 An expression cassette comprising the nucleic acid molecule of B1);
b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);
b4 A recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);
b5 A transgenic plant cell line comprising the nucleic acid molecule of B1) or a transgenic plant cell line comprising the expression cassette of B2);
b6 A transgenic plant tissue comprising the nucleic acid molecule of B1) or a transgenic plant tissue comprising the expression cassette of B2);
b7 A transgenic plant organ comprising the nucleic acid molecule of B1) or a transgenic plant organ comprising the expression cassette of B2);
b8 A nucleic acid molecule that promotes or enhances gene expression of a protein as described above;
b9 An expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing the nucleic acid molecule of B8).
In the above application, the nucleic acid molecule of B1) may be a gene encoding the protein as shown in B1), B2) or B3) below:
b1 A coding sequence of the coding chain is a cDNA molecule or a DNA molecule of a nucleotide of a sequence 2 in a sequence table;
b2 Nucleotide is a DNA molecule of a sequence 3 in a sequence table,
b3 A cDNA molecule or a DNA molecule which hybridizes with the cDNA or DNA molecule defined in b 2) and which codes for a protein having the same function.
The plant described above may be any of the following:
c1 Dicotyledonous plants;
d1 A monocotyledonous plant,
d2 A plant of the family Gramineae,
d3 A plant of the order Gramineae,
d4 A maize plant;
d5 Corn).
In the above biological material, the expression cassette containing a nucleic acid molecule of B2) refers to a DNA capable of expressing the protein of the above application in a host cell, and the DNA may include not only a promoter for promoting transcription of a gene encoding the protein but also a terminator for terminating transcription of the gene encoding the protein. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present application include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters, and inducible promoters.
Recombinant expression vectors containing the protein-encoding gene expression cassettes can be constructed using existing plant expression vectors. The plant expression vector comprises a binary agrobacterium vector, a vector which can be used for plant microprojectile bombardment and the like. Such as pAHC25, pWMB123, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Co.). The plant expression vector may also comprise the 3' -untranslated region of a foreign gene, i.e., comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The polyadenylation signal may direct the addition of polyadenylation to the 3 'end of the mRNA precursor and may function similarly to the 3' transcribed untranslated regions of Agrobacterium tumefaciens induction (Ti) plasmid genes (e.g., nopaline synthase gene Nos) and plant genes (e.g., soybean storage protein genes). When the gene of the present application is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancers may be ATG initiation codon or adjacent region initiation codon, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence.
In the above biological material, the recombinant microorganism may specifically be yeast, bacteria, algae and fungi.
In order to solve the technical problems, the application also provides a method for reducing the plant lodging rate, which comprises the steps of enhancing or increasing the expression level of the protein coding gene in a target plant or/and regulating the content of the protein or/and the activity of the protein, so as to reduce the plant lodging rate.
In the above method, the enhancement or improvement of the activity of the above-described protein in the target plant or/and the expression level of the above-described gene encoding the protein may be achieved by introducing the above-described gene encoding the protein into the target plant.
In the above method, the plant and/or plant of interest may be any of the following:
c1 Dicotyledonous plants;
d1 A monocotyledonous plant,
d2 A plant of the family Gramineae,
d3 A plant of the order Gramineae,
d4 A maize plant;
d5 Corn).
The proteins described above and/or the biological materials described above are also within the scope of the present application.
The protein provided by the application is obtained from corn, and is named MDR1 (Maize lodging Resistance 1) protein of protein kinase. Experiments prove that after the corn MDR1 gene is excessively expressed in corn, the content of lignin in the corn stalk part can be increased, and the potassium content of corn leaves can be regulated and controlled, so that the stalk bending force of the corn stalk is increased, the lodging rate of a corn plant under the condition of strong wind is reduced, and the lodging resistance of the corn plant is improved. The application has great application value in crop molecular improvement, and has important theoretical significance and practical significance for improving crop lodging resistance.
Drawings
FIG. 1 is a graph showing statistics of lodging rate and observation of lodging resistant phenotype of MDR1 overexpressing material. Detection of MDR1 expression in MDR1 overexpression Material (MDR 1OE-1 to MDR1 OE-4). ZmUBQ is an internal reference gene. And B, calculating the lodging rate statistics result of the MDR1 over-expressed material. SZ represents the upper village in beijing and AY represents the annan yang in henna. Lodging rate = lodging number/total number of plants. The total number of plants for each material was 40.C. Phenotypic observations of potted MDR1 overexpressing material (MDR 1 OE) under natural wind treatment in Beijing spring 2020. And D.2020, after-growth phenotype observation of MDR1 over-expression materials planted in Beijing Shangzhuang.
FIG. 2 shows the results of the measurement of the bending force of the stem in the male-pulling period of the MDR1 over-expression material. The measurement results are shown as mean ± standard error (n=20), t-test shows P <0.05.LK represents low potassium and HK represents high potassium.
FIG. 3 shows the results of the internode lignin content measurement and lignin synthesis gene expression test of the MDR1 over-expressed material. A. And (3) staining and observing the basal part of the MDR1 over-expression material in the male-pulling period by using phloroglucinol. LK represents low potassium and HK represents high potassium. The scale is 200. Mu.m. B. And (5) measuring lignin content of mechanical tissue of the spike node of the MDR1 over-expression material in the male-pulling period. The measurement results are shown as mean ± standard error (n=4), t-test shows P <0.05, P <0.01.C. And (3) detecting the expression of the internode lignin synthesis genes (ZmPAL 3, zmHCT and Zm4CL 1) of the MDR1 over-expression material and the mutant material in the male-withdrawal period. ZmUBQ is an internal reference gene. The detection results are shown as mean ± standard error (n=4), t-test shows P <0.01.
FIG. 4 shows the results of leaf and internode potassium content measurements for MDR1 overexpressing materials. Phenotypic observations of MDR1 overexpressing materials in 16 days to V6 stage of low-potassium and high-potassium hydroponic treatments. The pictures are shown as base third, fourth and fifth leaves. LK represents low potassium and HK represents high potassium. The scale is 15cm. And B, measuring the potassium content of the basal third leaf of the MDR1 overexpression material after 16 days to V6 phase of water culture treatment of low potassium and high potassium. The measurement results are shown as mean ± standard error (n=5), t-test shows P <0.01. And C.X ray fluorescence spectrometer detects the potassium ion content of the spike stem of the MDR1 over-expression material from the low-potassium treatment to the emasculation stage. The scale is 1cm. D. And (3) measuring the potassium content of the ear stem under the low-potassium and high-potassium potting treatment of the MDR1 overexpression material in the male-pulling period. The measurement results are shown as mean ± standard error (n=5), t-test shows P <0.01.
Detailed Description
The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The quantitative experiments in the following examples were all set up in triplicate and the results averaged.
The maize inbred line used in the embodiments of the present application is ND101 (PI 612589), and germplasm information can be queried for GRIN-Global https:// npgsweb.
The pBCXUN expression vector in the embodiment of the application is stored in the laboratory and related literature: qin YJ, wu WH, wang y.zmhak5 and ZmHAK1 function in k+ uptake and distribution in maize under low K + conditions.j intergr Plant biol.2019jun;61 (6) 691-705.Doi:10.1111/jipb.12756.Epub 2019Feb 1.PMID:30548401.
The corn material in the embodiment of the application is cultivated in two ways of pot culture and water culture:
in a potting system, seeds are sowed in vermiculite, germinated and grown in a precisely controlled underground greenhouse at 28 ℃ until a two-leaf period, and then the seeds are moved to a glass greenhouse (photoperiod: 14h (28+/-3 ℃) per 10h (23+/-3 ℃), humidity: 45%, and light intensity: 400 mu mol/m) 2 And/s) hardening off until the seedling reaches the three-leaf period, then selecting orderly and consistent materials, transplanting the materials into a flowerpot, and transplanting one plant into one pot. The method for mixing soil comprises the following stepsPreparing: 20-barrel potted plant requires a stirrer to mix evenly about 10 bags of domestic soil (15 kg in 1 bag), 1.25 bags of perlite (6 kg in 1 bag), 1 bag of vermiculite (8 kg in 1 bag) and 40g of Ca (OH) 2 About 8kg soil/barrel after sub-packaging. Pouring a nutrient solution for one time before transplanting, and taking the nutrient solution as a base fertilizer, wherein the nutrient solution is 2L/basin; nutrient solution is respectively applied once in the jointing period and the male pulling period. The experiment is designed into two treatment modes of high potassium (HK) and low potassium (LK). The low-potassium treatment is actually treatment without applying potash fertilizer because the pot substrate contains a certain amount of potassium ions. The formula of the potting nutrient solution is as follows:
table 1.HK potted plant nutrient solution formulation
Table 2.LK nutrient solution formulation for potted plants
In addition, 2000X (working solution concentration: 1. Mu. Mol/L MnSO) of a trace solution is required to be added to the nutrient solution 4 ,1μmol/L ZnSO 4 ·7H 2 O，0.01μmol/L CuSO 4 ·5H 2 O，0.005μmol/L(NH 4 ) 6 Mo 7 O 24 ·4H 2 O and 1. Mu. Mol/L H 3 BO 3 ): 0.5mL/kg soil; ferric salt 100× (working solution concentration: 0.1mmol/L Fe-EDTA): 10mL/kg of soil. And culturing the plants until the male pulling period for phenotype statistics.
In the water culture system, seeds are sown in vermiculite for germination at 28 ℃ for 5 days to one leaf and one heart, endosperm is removed, seedlings are slowly grown in HK nutrient solution for 2 days, HK (1.85 mM K) + ) And LK (0.03 mM K) + ) And (5) processing. The formula of the water planting nutrient solution is as follows:
table 3.Hk hydroponic nutrient solution formulation
Table 4.LK Water planting nutrient solution formula
Note that: the table shows the contents as working fluid concentrations.
In addition, the nutrient solution needs to be added with a trace amount of solution and ferric salt, and the concentration of the working solution is consistent with that of the potting nutrient solution. Finally, naOH is used for adjusting the pH value to 5.8-6.0. Corn cultures were performed using a 50L hydroponic cassette in a 28℃incubator. 8 seedlings are cultivated by 1 water culture box, and the water culture solution is replaced once in 2 days. Phenotypic observations were performed 14-16 days after treatment.
Example 1 construction and detection of maize MDR1 overexpressing Material
Construction of MDR1 overexpression Material
The amino acid sequence of MDR1 (Maize lodging Resistance 1) protein in the maize B73 is shown as a sequence 1 in a sequence table, the nucleotide sequence of an MDR1 gene coding chain is shown as a sequence 2 in the sequence table, and the genomic DNA sequence is shown as a sequence 3 in the sequence table.
Sequence 1:
MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQEWTDDLVRLRERYPWLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGSTLYTHLLKSLEAQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGFWQLDFSLPVPPRPVLPVTEI；
sequence 2: (5'-3')
ATGAGCCCAGAACGACGCCCGGCCGACATCCGCCGTGCCACCGAGGCGGACATGCCGGCGGTCTGCACCATCGTCAACCACTACATCGAGACAAGCACGGTCAACTTCCGTACCGAGCCGCAGGAACCGCAGGAGTGGACGGACGACCTCGTCCGTCTGCGGGAGCGCTATCCCTGGCTCGTCGCCGAGGTGGACGGCGAGGTCGCCGGCATCGCCTACGCGGGCCCCTGGAAGGCACGCAACGCCTACGACTGGACGGCCGAGTCGACCGTGTACGTCTCCCCCCGCCACCAGCGGACGGGACTGGGCTCCACGCTCTACACCCACCTGCTGAAGTCCCTGGAGGCACAGGGCTTCAAGAGCGTGGTCGCTGTCATCGGGCTGCCCAACGACCCGAGCGTGCGCATGCACGAGGCGCTCGGATATGCCCCCCGCGGCATGCTGCGGGCGGCCGGCTTCAAGCACGGGAACTGGCATGACGTGGGTTTCTGGCAGCTGGACTTCAGCCTGCCGGTACCGCCCCGTCCGGTCCTGCCCGTCACCGAGATTTGA。
Sequence 3: (5'-3')
GATTTTAGTACATGTGTGGGCTTCTTCGCTGTTTTTTAGTTGGGTGCACATAGCCCAAGTATAAAGGGACCGAAGAACTCTAAGGTTATGGCCAATAAATTATATATATATATATATATATATATATAACCATGTAAAAATATTATATTATATTGTAGAATGCACCGTTTATAGATTATTTATAAGGTCCCGTTTGATTTAGGGTGACTAAAGTTCTAAAGCTTAGTCACTTTAGTCCCTAAAGAAGTAAATATGGTGACTAAAATAAGGTGACTAAACTTTAGTTCTTTAGTCAACAAGGGGTGACTAAAGTATAATTTTTACCGTATTAACCCTCTCTATTTTCTTGTTACAACAAACATATACTATTTAATAAAGGTAATATAGTCATTATTCACAGTAATTAATGCTCTTTAGTCCTGTTTAGTCACTGAAAACAAATGGGACCAAACGGTGTACTTTAGCGACTAAACTTTTCACTAAAGTCTAGTCTAGTGACTACAAAACAAAGCCTTATTAACGCAGATTACATTATAAAATGAAGTTTGTTGTGGTTCTAGATAGGCTAATAAAAAAGCGGATAAAGGAGAGGAGCGGGCACGACGGCTGCAGGAGACCCGTATAAATACGATCTGGTTTCCGTTCCCAATCCCCTTCTTCCCCGACTCGCCCGGCGCTGAATAAGATTCCCCTGTATTCTGTTCCAGAGATGAGCTGATCGAGCAGCCCGTCGCCAAGCCAACCGGACATGGTGAGCGACGGCGCGCGCGGGCTGCTGGGCGCGTACGAGCTCGGGCGGACGCTCGGGGAGGGCAACTTCGGGAAGGTGAAGCAGGCGCGGCACCGCGGCAGCGGCGGGCAGTTTGCCGTGAAGATCATGGAGCGCGCCAGGGTGCTGTCGCAGCGCGCGGACGAGCAGATATGCCGCGAGATCGCCACGCTCAAGCTGCTCGCGCACCCCAACGTCGTCCGCCTCCACGAGGTCGGTGTCGGTCCGCTCGTCTTCCATCTCCGAATTCCTAGATTTGTTAACATAGATTCGTTGCGTGTTGAGACGCCTTCGCCGTTCCTAGATTTGCTTCTCGAAAATTCCACCCATCTCTACCCCAGTTGTTTTTTTTTTTGATCGATTTGAGTATTACTGGTAAGTATCGCAGTTTTTTTGATCGATTTGCCTGGCCGCAGCTCAGCTGCAAGCCTGCACTGCAACTTGGCCACTGTCCACTGCTGCAAGACTTTTGTCTACTGGCTCTCGCTCGTCTTCGTCTACCGCACGTGCACGGTGCACCACACCACCCTCTCAGCCACAATTTTCACCCATACAAATTTTGGAACGTAAAAGTCCAGACTTTCAATACTTAGTATAATTACTAATTAGTACTACTAGCATGTACACTCTACTTAGCAAAGGTTGAGCCCATAGATCTGGACAGCGATAAATTAATAATATTTGTAAATATTGTCGATGTCTTTCTGTACTTTTTCGGTCTCGGGATAACGACGCACCTCACGTATCAGAGAGTCAGCATTTTTAAAAAAGATAATATTAACTTATATGTAAAGAAATTCTAGTATAAGTATTTATATCTTCGAATAAATTTATTATTAAATTTATTATCGAATAAATCGATTATTTTTTATATATAATTTTAAAACATTTGTCTTCTCGGTGTACTTGCTACTTTTTTCGGAATGAGGGAGAGTAGCAGTAGTGAATCAATGCTGCTGGTGCACATGGCATGCACACCCGTTGGCAGAGACAGGCGTGTGGGTCCGCCTCGGCCTATCCCAGTCCCACCATAGTGAGGCTGAGCTGGTGGACACGGTGCTCCATGTCGCGCTGCTCCGGGTCCACCAATTATTGGCACGACCCACCCAACTACCCAAGTAGTAGTAGTATACAATTATTGCTTAATTAATCCAACTTGCAACACCCCAGTGATTGTAGTGTTTGTTACAAAACATGTTACAATGTGATTAAATCCCTCCATTTGACAATTACTTCCCAAGGGACACACCCATTGGAAAACACTGTTTAATCTTGTATATAACGGAACCCCCTTTGCAATAAAGACACACAACAGTCACTTTCTCTCGGTTTCAAATCTCTTTGCCACTGAGTTTCAACACGTTATCAGCACTGCTCAAAGAAAATAGAGGAAGAAGAAGAACACTTGTTGTTCTTGACAAGAACAGAGGCAGCGGTCTCCAAATCAACAGGAAAGGTATGGAGCAAACCATTTCCCTTCACATCCGTCGTATGCGTGAAGTTCTCCATGTTGTTGATGACACACTGTTGGAGGCATTCGCTGCTGGGCAAGACGCAGCGATCCCGCGGATGAACG
TCATCCCTGAACGCCGTGTTCACGTGCAAAATGTGCACATTTGTGTGATCTACGGGTGG
ACAACAGTGGTGCCGCTGCCGGTGGTTGAGTGTGGCCTCCCCACTGCGTCCCCGTCACC
ACCGGTGCCGATGGGAGACGCGGAGTCGTCGGCAATCGCACCGATGGACGTCGATCTG
GAGGCACCACCGCCTCCTCCACCCACTGCAGCGGCAGTTGGTCGTCGTTCCCCAACTCC
GCCGGTGTCGGTGGTCGTTGGGACCATCGCTGCTCGTCATGGACAGCCCCCTCGCACCT
TCGACGCCCCGCGCGAACGTGTCATCTTCACCGGCCACCGCCCAAATGGGGTGGCCCC
ATCTTCCTCCTCCAATGGGAGGGCCTAATGGCCCTCGATCCGGTCGGTCGCGGTTGCCA
CCACGACGGCCGGTTGGGGGAGGGGAGCAGCAAGGTCACGGACGTGGCCCCTGCGCG
GCGCGGCTCGGAGCCTTCCCGACGAGGGTCCTCGGCCCGGCGCGCGACCGCGGTCGGG
CGCGGCCCTAGCGATGGGTGCGGTCGGCCACTGGCCCAGGCGTGGGTGCCCCCCGCCC
CCTCCTTGGCGCGACCGACTCCCCGGCGCGCGGACGCGCTGCCACGGCCACGGACGCG
GCCCTAGCGAACGGCCAGCCGGCCGGCACAGCAACGGCGCGGTCGTGGCGGCCCTGA
CGAGAAGGGCGCGGCCGGATCCGCCCCCCTGGCGGCGGCGCGTCCGGCCAGCCGTGG
GTGCTGCTAGACCTGGCCAGGGCGCGACAAACAGCGAACACGGGTGCAGCCCCTCTAC
GCAGGCGAGGGCCCGGTCGGATCCGCCCTCGGGAGGCATACCTGCGGCTGATGGGCAC
TATTCCTAGCGGCCGGCGCTCAGGCATGGCGATGGCGGCTCTCTGGTGGCTAGGGCGG
TTAGGGTTGCAAACCCTAGCCGCAACCCTAACCGCCCCCTTTTCTAATAGGCACCATGG
CCTCCTGGTGGGACGCCTGGGCCGACGTGCTAGCCACCAGGCCGCTGGCTGGCCCGCC
TGGCAGGCCGCCTGGGCTGGTCCCATGGGCCCAGAATGGTCTGTTTATCATTTTTAAAT
TCAGAGTTTTAGTATAAGACTCTGTTTTATATGCACATTCAGTCTTAATTTGTGTTAGGA
TGTCTAAATTTGGCATTTAGACTGCAAATTGCTATATATATTGTACATATTTGTCTGTTT
TATTATGAATATAGCAGACTTTAGTTCGTGAATTATTTTTGTTTTTACATTTATAATTCA
CATCTCTACTTGCTATTATTTTCGCATTCTTTTATGTTTGCAATCAAATATAGCAATTAT
TAAGTTCAAACTTAATAATATTCATGCAAAATAAAATTACATTATTTTGGAATTAGAGC
ATAGAGTGATTAGAGTCCTAAGCACTGGCCACACTGAATTTTTTATAGAGTTTAGTAGC
CTAGAAACCGTGCTTCTGGCAGCATTTTAAATGACTATCTCTATGAGGTCTACATCTTT
CGGGATGATTACCTATACGTGCTATCCAAAGTAACAGGATATGGAGTTCTTGAATATTA
TTTTGATAATTAGTCGAGCATGGGTAGTGGAGACCTAAGCATTGACTGCGGTTTGTTCA
TTCATGAACTTATCAGCCCTGAGACCGTGCTTTTAGGCAGCAAGTTTCTTGCCCACTAC
TAGGAGATCTACCTCATTAATTTGAGGATTATCTATAAATTGTTCACTAATATTCAAAT
TATTTTTTTTCAATTGGAAGGTTTGGAACCTTTAGGCAACTCATATCACATGTAATTTTA
AGGTTTTTGCATTTGAGCTTACAACATCACCATTGTGACGATTAATTTATACATAAATT
AATGGATGTCCATGGTCATGACTGTAGGGACATGTCAACTAAAGAGTTCGAGGAACTC
ACCCTTGATGGGCATAATTACCCAACTTGGGCTTCAGATATTGAAATAACTTTTGTTTC
TCGTGGGATCATTGATGCTATAGCAGCACTTATCACTGGTACTGCTCTAGTTAATGAAG
TTAAAAAGAACACTGCACTTTTCCTTTTTAGGCTTTACATCCATAAGGATCTGAAGCAG
GAGTACCTTATGGAAAGATGCCCTCATAACTTTTGGAAAGCTCTTAAAGAGCGCTATG
AACAGTAGAAGGAACTCATATGGCACTCTGCCAACCATGAGTGGAATCACCTTCGCCT
GCAAGATTTTAAATCTGTTGCAGAATATAACCATGCTCTTCATAGTATTTTGACCATCG
CTGGACGCGATGCGGTGATAATTGGCTCTGTATGAGCAACTATTACGCTCCCCATGGGT
ACTGAAATTGTTATCGAGGATGCATTATTGTATCCTGATTCCACTCATACCCTCCTAAG
TTTTAGAGATATCCGGCAAAATAGATTTTATATTGAAAACCATGATAAAAATCAAGAA
GAGTTTCTCTTTCTCTTCTTGACCAAGCCGAACAAATATGGCAAACACATATGCGAGAA
AATTCCATCACTCACGTCTGGGTTGTACTATACATACATTAAGACCATTGCACATGTTG
CATATAAGGTGATTTTCCAAAATGTTGATCCATTCTAGGTTTGGCATGATCGACTTGGT
CATCCAGCCATACGGATGATGAGAAAAATCACAGGGAATTCTATGGTCGTAATTTCCC
TACAAATTTTCCCAAATCTAAAGATTTTATTTGCACTGCATGTGCAACTCGGAAACTGA
TTTTGAGACCATCACATCTCAAAATTAAAGCTGAACCGCTTAAATTCTTTGAAAGAATT
CAAGGAGATATTTGTGGGCCAATTACACCAACTTCTGGCCCGTTCAGATACTTCATGGT
ATTAATTGACGCATCTACAAGAAGGTCACATGTGTGTTTACTATCAACACATAACCATG
CATTTGCCAAGATAATGTCTCAAATTATCAAGTTAAAGGCAAATTTTCCTGAACATCGA
ATTAATTCAATCTGGATGGACAATATTGCTGAATTTACATCTCAGGCATTCAATGATTA
TTGTATGGCGCTGGGTATTCAAGTACAACACTCGGTACCATATGTCCACACCCAGAATG
GTTTGGTTGAGTCACTAATCAAGAGGATTAAACTCATTGCAAGGCCATTATTATTGATG
AATTGCAAATCACCTTCGTCGTGTTGGGGTCATGCAATTCTGCACGCTGCAGACCTTAT
ACAACTTCGACCTACTGCATATCATTTTACTTCCCAATACAAATGGTAGGTGGAAATCC
TCCAAGTATTTCTCATTTGCGTAAATTTGGATGTTGTGTATACATTCTAATTTCACCACC
TTAGAGAACAACCATGGGCCCACACAGAAAAGTGGGGATCTATGTGGGATTTCAATCT
CCGTCGATCATTAAGTACTTAGAACCCTTGACAGGGGATCTATTTACGGCTCGGTTTGC
TGATTCTATTTTTGATGAGGACATTTCCCGACATTAGGGGGAGACTTTAAGTACCCAAA
GGAATGTCAAGAAATAAATTGGAATACTAAATGTGTTCCAGGTACCGATCCACATACT
ACAGAAACTGAACTGCAAGTTCAGAAGATTATACATTTGCAAAGACTTGCAAATGAAC
TACCAGATGCATTCACTAATTATAAAGGTGTCACTAAATCATTCATTCCTGCTAGGAAT
GCACCAGAATGAGTGGAAATACCGAATAAACCACTCAACTCATAAAGAGGAGAAGTA
TGGTGAATAAACGCTATTCTGTTGCTAAGAAGCAAAGAACAATAATGAATGCAAACAG
ACATCAAGAGGATACAATGTATCAAGTGGATTGTGATGATCCGCGACCTAGCTCGGAT
GAGCACATTGTCGAGACTAGGACATCGGAAAACCCTAGACACATCAATTTGGGAAATT
ATGATGAGTCTCTATAGGGTGATGAAATTGCCATCAACTATGTTGAGACCGGTGAAAC
CTATGATAGAAAAGCTACCAATGTCGATATATATTTCTCCGAGAAAATTGCTAAGGAC
CTTTAAAATGATCTAGATCCTAAGACCATGGCAGAGTGCACTAAGCGCTCGGATTGGA
TCAAGTGGAAGGCAGCAATTGAAGCGGAGTTAGCCTCGCTTTACAAAAGAGAAGTTTT
CTCGGCTGTAATGCCTACACCACGTAATATCTTCCCTGTGGGATATAAGTGGGTTTTCA
TTCGAAAACAAAATGAAAATGTAGGTAGTGAGATATGAAGAAAGGCTTGTAGCACAA
GCCACAAGGGTTTACGCAAAGACCCGGCGTTGATTTCAAATTCGTTTATCTTTGCAGTT
GATGGACGTACAAATTCCGATATGAGTGCAATAACATTCCGATATTGAATATCTTTGGT
AGTACAAATTCGTTTATCTTTGTAGTTGATGGACGTAGTGACCGCATATCTCTATGGGT
CACTGGATTCTGATATATACATGAAAGTTCCTGATGGAATAGATATACCGAATCCAAA
GGCAAAACACAACATGTATTACGTAAAGTTGCAGAAGTCATTATATGGCTTAAAACAA
TCGGGCAGAATGTGGTACAACCGATTGAGTGGATTCCTTTTACGTAAAGGATACACTA
AAAATGATGATTGCCCGTGCGTCTTCATCAAAAGATCCAAAGTGGGATTCTGTATCATA
TCGATTTATATTGATGACCTCAACATCATAGTAAATAAACTTGATATTGATGAAGCACA
TCATCATTTAACGACGGAATTTGAGATGAAGGATTTGGGTAAAACCAAATTTTGCTTAG
ATCTTCAACTTGAGCATTTACCTTCTGAAATCCTAGTACATCAAAGTTCAAATACCCAA
AAGATATTGGAAAAGTTCAATATGAATATGTCTTATCCTTGAAAGACTCCTATGGTGGT
CAGATTTTTGGATTTAGAGAAAGATCCATTCAGACCACGGGATGATGAAGAATAGATA
TTGGGACCTGAGTTCCCATATCTCAGTGTCGTTGGTGCATTGATGTATTTTGCTAACAA
TACCCGGCAGGATATTGCTTTTGCAGTAAATTTGCTAGCAAGACACAGCTCTGCTCTAA
TAAAACGACACTGGGCTGGCATTAAGAATATTTTACTATATCTAAATGGCACAAAGGA
TCTAGGACTTTTTTACAAAAAGAAGTAATGATCCTAGTTTAATTGGGTATACCGATGCT
ATTTATCTGATCCCCACAATGACATATCGCAAACATGATTCGTATTTTTAAAAGGTGGA
ACACTGTTTAATCTTGTATATAAGGGAATCCCCTCTGCAATAAAGATACACAACAGTCA
CTTTCTCGCTTTCTCTCTGTCTCAAATCTCTTTGCCACTGAGTTTCAAAAGTGTTCCTGA
CCGTGTGTTCTTGATCCAGGTGGCTGCCAGCAAGACGAAGATCTACATGGTGCTTGAG
CTCGTCAACGGCGGCGAGCTGCTGGACAGGATTGTAAGCCGCCCCGGCCGTTCGTTCTT
CCTCCCTTGTGATCAGTCAGGCTACACTCCTTGTCCGATGCCGTTACCACTCCCATACG
CGTCTGATCCCCCTCCCATTGCTTCCCTTTTGCTGCAACGGCAACTGACCAGCAGGCGG
CCAGTGAGGGAAAGCTCCCGGAGCAGGAGGCAAGGAGGCTCTTCCAGCAGCTGGTTG
ATGGTGTAAGCTACTGCCATGAAAAGGGCGTCTGTCACAGAGACCTCAAGGTGCAGTG
CCGGCCGGACGTTCCTCATCCCGCTCCGCTCCTATCAGTTAAGTTTTGCTGTGTCCGTG
GCCCTGGTCCTTTCCCCTAATCCACGTTCCACCTGTTCACAGCTGGAGAACGTTCTTGTC
GACCGGAAGGGAAACATCAAGATCTCTGATTTCGGGCTCAGCGCTTTACCGCAACATC
TCGGGGTAGGTACTACTATATGATAAACAGTTCTCTGTCAAATGGTTTTCGCAACATCA
AAATCGCTCATCGTGCTGCCTGATGTCTTTCTTTCTTTCTTACTTACTTCCAGAACGATG
GGTTGCTGCACACGACCTGCGGTAGCCCCAACTACATTGCCCCTGAGGTGAAGAAATC
CTGACTACTCTACTCACTCACTCATCACTCCCAGGCGGCCAAACAGCTCCCCAGGTGCT
AATTCCTTGTGCCGAATGCCTGCAGGTTCTGCAGAACAGAGGGTACGACGGGTCGCTA
TCGGACATCTGGTCATGCGGGGTGATTCTCTACGTGATGCTCGTAGGGCACCTCCCGTT
CGACGACCGGAACATCGTCGTTCTCTACCAGAAGGTACTGGATCAATCAGATAAATCG
TTTTGTAGCTCTTGCTACTTTACAATTTAATTTGCCGGGTCTAGATTTTCAAGGGCGATG
CTCAGATCCCGGAGTGGCTCTCTCCCGGCGCACGGAACCTTCTCCGGAGGATCCTTGAG
CCCGACCCGGCGGAGCGGATCGCCATGGCAGAGATCAAGGCGCACCCATGGTTCCAGG
AGCACTACGTCCCTGTCCTTCCTCCGTACGACGACGACGACGACCACGATTCAGTGAA
GCAAATCGGTGCGGGTAGTAAAACCACTCATCACATCAACGCGTTTCAGTTGATCGGA
ATGGCGTCGTCCCTAGACCTCTCAGGGTTTTTTGAGGAAGAGGTACGGTGTGGTGTGTG
GACAACATCTTGGGAACTTCCTTTTCTTTTTTTTCTTTTTTTTTCCTCCTCCAGCACAGCG
ATTTGCCTCTTGTGTGCTCAAAACAATTCGCTGCACTACTACTGATACATAGATCACAC
ACTGACTGAATTGAGCTCAGAGTAAACTGTTCTTCTAACTCTCTGCTGCAGGACGTGTC
CCAAAGGAAGATCAGGTTCACGTCCGCACAACCGCCTGAGGATTTGTTCGCCGAGATC
GAACGCTCTGCGACTGACATGGGCTTCCATGTTCACAGAGGGCATAGTAAGGTCAGTC
AGTACAGACTTTTCCATCGACCGCAGCACTGCCATTACATTCAACGTTGACGTGTGTGG
CCGGTTCTTCAGCTTAGGCTGACGCGAAACTGCGACCGGTCGAAGAATCAGAAGAACC
CTATGTCGTCGTCGTCGTCGTCGTTTCTAGTCTGCACCGAGGTAAAACGCAGCAATTAA
ACTGCATGCACCCCCAGGTTTTTGAGCTAACGATGGAGTGCTGTGCCATCTGTGATCCC
AGGTTTTTGAGCTTAGCCCCTCTTTGTATGTCGTGGAGCTCAACAAGTCCCATGGCGAC
CCTGCACTGTACAGGCAGGTAAGAACAGCTGCATGCATGCACTTGTCAATGACTCACT
GCCATGGCTAGCAGTTCCAGCGACTAAGTGGTCCGCCTGCTTCTTGAAACGTGCTCCGC
AGCTCTGTCAGAGGATCTGCGGTGACCTAGGTGTGCTCGAGATGGACCAGATCTTTGG
GACGAGGCCGCCGGTCGCCGACGATCTCGCGGGCTTGGCCTTGGCGTTGGAGAGACGA
TCTGCGACGCCTTTGGTTGCGTTGTGACGAAAGGCTGCCCCCGCCGGCCCCCCTGACCG
TGATGTTTGTAGCGTCAGATTATTTAGTGACGACGAAGCAGTTCAGCTGCAACGCAGCT
CGGAACGGTTGTGTGATCCCTCCCAGAAATAGAGGACAAGGTAAAAGAGTGTAGCCGT
ACACGTGTAGGATCATGCTCCAATGTAAATAAAGTGGTAGTAGCCACCGATGTAAAAC
TGAATGTAAAATCTAAGAAATAAATCGTTTATCCAGTACACAGAAGCTCTATAGTGGT
GGTACTAGAGCTATTTGTACTAACGTTTTTCAGTGCCACCAGGGTCAGTAGAAATCGTC
CTTTTCACTGACGGTTGTTTTGCAACTGTCAGTGCAAATAACATTAGCACTGGCAGTCT
GCTTAAGAAAACCGCCTGTGGAAACAAGATTTTCACTGGCGGTTTTCTAAAGCAAACC
GCCAGTAGAAATAATATTAGCACTAGCGGGCTGCTTAAGAAAACCGTCTGTGGAAACA
AGATTTTCACTGGCGGTTTTCTTAAATAGCCCGCCAGTGGAAATAACATTAGCACTGTC
GGGCTGCTTAAGAAAACCACCTGTGGAAACAATATTTTCACTTGTAGTTTTCTTAAGCG
GTCCGACAGTGGAAATAACACAAGCACTGGCGGGGTACTTAATAAAATCGTATCTGAA
AACAAAATTTTCACTGACGGTTTTCTTAAGTGGTGTAACACCTAAAATTATGATTTTTG
AAATTAGTAAAAGAAATCAAAGAAAAGTGTCCTAATAAATAATTATAATATGGATTTT
TTTATGACAGTTAAAATAATAAGTTTAGTTATAAATTTCAAGAAATAATGAGAATAGCTTCAATTTATTAGAATGGATTAACCCCTTCTAAGGCTA。
The overexpressing material MDR1OE (MDR 1OE-1 to MDR1 OE-4) maize seeds for obtaining the maize MDR1 gene were constructed using the pBCXUN expression vector saved in this experiment (Qin YJ, wu WH, wang Y.ZmHAK5 and ZmHAK1 function in K+ uptake and distribution in maize under low K +conditions.J Integr Plant biol.2019Jun;61 (6): 691-705.Doi:10.1111/jipb.12756.Epub 2019Feb 1.PMID:30548401).
RNA is extracted from the B73 leaves of corn, and cDNA is obtained by reverse transcription by taking the obtained RNA as a template. Using the obtained cDNA as a template, primer F:5'-ATGGTGAGCGACGGC-3' and R:5'-TCACAACGCA ACCAAAGG-3' PCR amplification is carried out to obtain a PCR amplification product, and sequencing is carried out on the PCR amplification product, wherein the sequencing result shows that the PCR product contains the CDS sequence of the MDR1 gene shown as the sequence 2 in the sequence table.
And (3) recovering and purifying the amplified PCR amplified product to obtain a recovered and purified product. The recovered and purified product is inserted into XcmI restriction enzyme cutting site of the expression vector pBCXUN by means of TA cloning to obtain recombinant expression vector pBCXUN-MDR1, and the XcmI restriction enzyme cutting can form 2T tails. The pBCXUN vector insert is about 5227bp in length from the Left Border (LB) to the Right Border (RB) of the T-DNA, wherein the target gene expression cassette (pUbi promoter+XcmI:: ccdB:: xcmI+NOS terminator) is about 2977bp in length, and the marker gene expression cassette (35S promoter+Bar gene+CaMV ployA terminator) is about 1455bp in length.
Competent cells of Agrobacterium EHA105 transformed with the recombinant expression vector pBCXUN-MDR1 were cultured on YEP medium (containing kanamycin 50 mg/L) at 28℃for two days, and positive clones were selected using the primer F:5'-ATGGTGAGCGACGGC-3' and-R: 5'-TCACAACGCA ACCAAAGG-3' PCR was performed (product size 1392 bp). The positive bacterial liquid obtained by PCR identification is named recombinant agrobacterium EHA105/pBCXUN/MDR1 and is preserved at-80 ℃.
Transformation of maize inbred Material ND101 with recombinant Agrobacterium EHA105/pBCXUN/MDR1 to obtain T 0 The generation MDR1 transgene over-expresses corn material, and a PCR amplification method of screening marker Bar gene (primer Bar-F: GAAGGCACGCAACGCCTACGA; bar-R: CCAGAAACCCACGTCATGCCA, target fragment about 262 bp) is used for detecting positive plants; will T 0 T obtained by harvesting transgenic overexpressed corn material of generation MDR1 1 Planting and identifying the transgenic overexpressed corn material of the MDR1 generation until T is obtained 3 Seeds of the generation MDR1 transgenic over-expression maize material MDR1OE (MDR 1OE-1 to MDR1 OE-4).
Detection of MDR1 overexpressing Material
Trizol (Sigma) method for extracting T growing to V3 phase 3 Transgenic overexpressing maize material for generation MDR1 (MDR 1OE-1 to MDR1 OE-4) and corresponding wild maize inbred ND101Total RNA from the whole plant was checked for RNA integrity by agarose gel electrophoresis. cDNA was synthesized as a template according to the instructions of a reverse transcription kit (Semerle Feishmania technologies). MDR1-Primer 1 and MDR1-Primer 2 primers (10. Mu.M) were diluted 10-fold and mixed, and a reverse transcribed template was diluted 5-fold and subjected to Real-time PCR. The Real-time PCR system is: 10. Mu.L SYBR GREEN, 2. Mu.L primer, 1. Mu.L template, 7. Mu.L ddH 2 O, total 20. Mu.L. After confirming that the dissolution curve of the primer is unimodal, the primer is put on a machine, and finally 2 is utilized -△△CT And processing the data. The reference gene is ZmUBQ. The relevant primer sequences are as follows:
MDR1-Primer 1：5'-CGGTGCGGGTAGTAAAACCA-3'；
MDR1-Primer 2：5'-ATGGAAGCCCATGTCAGTCG-3'；
ZmUBQ-Primer 1：5'-CTGGTGCCCTCTCCATATGG-3'；
ZmUBQ-Primer 2：5'-CAACACTGACACGACTCATGACA-3'。
quantitative detection results showed that the MDR1 gene was over-expressed in all of the 4 MDR1 over-expression lines (MDR 1OE-1 to MDR1 OE-4) relative to the wild-type maize inbred line ND101 (FIG. 1A).
Example 2 functional verification and phenotypic detection of MDR1 protein
Respectively selfing the corn MDR1 over-expression materials (MDR 1OE-1 to MDR1 OE-4) for three generations to obtain T 3 Substituted MDR1 over-expression material. The MDR1 over-expression material strain (MDR 1OE-1 to MDR1 OE-4) T 3 The generation plants and the wild control corn inbred line ND101 are respectively planted in a south greenhouse (comprising two treatments of high potassium and low potassium of potted plants) of China university, a Beijing Shangzhuang test station (only high potassium treatment of a field), a Henan Anyang test station (only high potassium treatment of a field) and a Hebei 280955 state test station (comprising two treatments of high potassium and low potassium of a field) in four places, and the lodging resistance characters of the materials are observed and counted.
1. Planting conditions and treatments
The planting condition of corn materials in the field: the row length is 2.5m, the plant spacing is 0.25m, and the row spacing is 0.5m. Coating the seeds before sowing, sowing single or double seeds, and growing seedlings to 10 plants in each row in the period from the emergence of seedlings to V3. Three replicates were set for each treatment in the Beijing Shanghai laboratory and Hebei laboratory, 280954, each replicate including 20 individuals. The field test at Henan Anyang test station was an initial phenotype screening experiment with a total plant number of 40 per material.
The fertilization conditions of the high-potassium land block are as follows:
TABLE 5 fertilizing formulation for high-potassium plots
The fertilizer application of the low-potassium plots was performed by applying urea (202.5 kg/ha) and calcium superphosphate (240 kg/ha) only, without applying potassium sulfate. The nutrition treatment is mainly to fertilize before planting, to fertilize during the jointing period and to supplement fertilizer before emasculation.
The high and low potassium potting treatments of the south greenhouse of the university of china agriculture each set 5 replicates, each comprising 1 individual plant.
2. Phenotypic observations and statistics
Phenotypic observations and statistics include lodging resistant phenotypic observations, lodging rate statistics and stalk bending force determinations. Wherein lodging rate = lodging number/total number of plants; the stalk bending force is the force required for bending a maize plant at a height (from bottom to top) of 50cm by a fixed angle of 20 DEG when the maize plant grows to the male stage, and is measured using a plant stalk strength measuring instrument (SY-S03, shijia Shi sub-family technology Co., ltd.).
Under the natural wind treatment in Beijing spring in 2020, lodging-resistant phenotype observation is carried out on the MDR1 overexpression material planted in a pot culture and the control inbred line ND101, and the phenotype of the MDR1 overexpression material planted in Beijing Shangzhuang in 2020 in later growth period is observed.
The phenotypic observation experiment results show that: the field screening of the experimental stations of the Beijing Shangzhuang (represented by SZ in FIG. 1B) and Henan Anyang (represented by AY in FIG. 1B) of 2016 found that the lodging rate of the four strains of MDR1 over-expression material (MDR 1OE-1 to MDR1 OE-4) was significantly lower than that of the control material ND101. The result shows that the MDR1 over-expression material has better lodging resistance. Furthermore, after the strong wind weather in Beijing spring in 2020, the two over-expressed strains of MDR1 (MDR 1OE-1 and MDR1OE-2 of FIG. 1C) exhibited a better lodging resistant phenotype under potting conditions than the control ND101. Meanwhile, the MDR1 over-expression material is planted in a Beijing Shangzhuang experiment station, and compared with the control material ND101, the MDR1 over-expression material (the MDR1OE-1 and the MDR1OE-2 in the D in the figure 1) shows good stalk standing performance. Both potting and field experiments showed that MDR1 was involved in regulating maize lodging resistance (figure 1).
After planting MDR1 overexpressing materials in the field of 2020, 280950, in plots with normal potassium application (high potassium plot treatment, HK) and no potassium application (low potassium plot treatment, LK), the stalk bending force was measured during the emasculation phase and found to be significantly higher for the MDR1 overexpressing materials (MDR 1OE-1 and MDR1OE-2 in FIG. 2) than for the control ND101 (P < 0.05). This indicates that the MDR1 overexpressing material has higher stalk strength (fig. 2).
Example 3 lignin content detection and analysis
T of maize MDR1 over-expression line 1 (MDR 1 OE-1) grown to the emasculation stage grown in southern greenhouse at agricultural university of China by potting planting in 2020 3 T of the generation plants, line 2 (MDR 1 OE-2) 3 Phloroglucinol staining was performed on mechanical tissues between the basal fourth internode of the generation plants and wild control maize inbred line ND101 plants to observe lignin content.
1. Lignin staining
(1) And taking the middle part of the 4 th internode of the basal part after the corn plants grow to the emasculation stage. The resulting mixture was sliced into 60 μm pieces by a paraffin microtome and temporarily put into water.
(2) The sections were picked with forceps and placed on slides, and the material was spread and blotted. 5% phloroglucinol dye solution (0.5 g phloroglucinol is dissolved in 95% ethanol, the volume is fixed to 10mL, and the solution is preserved in dark place) is added dropwise, and the solution is dyed for 2min. Then dropwise adding an equal volume of concentrated hydrochloric acid, and standing at room temperature for 2min. Finally, 40% glycerol is dripped, a cover glass is covered, and a color CCD fluorescence microscope is used for observation and photographing.
The experimental results are shown in fig. 3, and the dyeing of the mechanical tissue is deeper under normal potassium application (HK in fig. 3, high potassium plot treatment) compared to the treatment without potassium fertilizer (LK in fig. 3, a), indicating more accumulated lignin. In addition, the over-expressed material (MDR 1OE-1 and MDR1OE-2 of FIG. 3A) was significantly darker than the control material (ND 101 of FIG. 3A), so that over-expression of the MDR1 gene favors the accumulation of corn stalk lignin.
2. Lignin content determination
(1) About 10mg of a sample which normally grows to the internode position of the corncob in the male stage is weighed, 1mL of freshly prepared 25% acetyl bromide (v/v) and 40 mu L perchloric acid are added, the mixture is fully and uniformly mixed, the mixture is subjected to water bath at 80 ℃ for 40min, and the mixture is vibrated for 1 time every 10min, and no sample is added as a blank control.
(2) After naturally cooling to room temperature, 400. Mu.L of 2M NaOH and 70. Mu.L of 0.5M hydroxylamine hydrochloride are added, and the mixture is stirred and mixed uniformly.
(3) The supernatant was diluted 40 times and absorbance at 280nm was measured by a UV plate, and the absorbance was recorded as A blank tube and A measurement tube, respectively. Δa=a measurement-a blank value.
(4) The acetyl bromolignin content was calculated according to the following formula:
lignin (mg/g dry weight) = 0.0735 × (Δa-0.0068)/w×t
Wherein, W: sample mass in g; t: dilution factor.
The results of lignin content measurements indicate that the MDR1 overexpressing material (MDR 1OE-1 and MDR1OE-2 of FIG. 3B) has lignin content significantly higher than control ND101, both under high potassium (HK) and low potassium (LK) treatment conditions. This result demonstrates that MDR1 participates in regulating lignin synthesis process in stalks, and that overexpression of MDR1 can increase lignin content in corn stalks.
3. Lignin synthesis gene expression assay
The expression levels of lignin synthesis key genes ZmPAL3, zmHCT and Zm4CL1 in the ear internode of the MDR1 over-expression material and the control ND101 material are detected and analyzed.
Extracting total RNA between the MDR1 over-expression material grown to the male-withdrawal stage and the ear node of wild type control ND101 by using a universal plant total RNA extraction kit (RP 3301) of Baitaike company, and checking the completion of RNA by agarose gel electrophoresisIntegrity. cDNA was synthesized according to the instructions of the reverse transcription kit (Semerle Feishmania technologies). The Primer 1 and Primer 2 (10. Mu.M) were diluted 10-fold and mixed, and the template after reverse transcription was diluted 5-fold and subjected to Real-time PCR. The Real-time PCR system is: 10. Mu.L SYBR GREEN, 2. Mu.L primer, 1. Mu.L template, 7. Mu.L ddH 2 O, total 20. Mu.L. After confirming that the dissolution curve of the primer is unimodal, the primer is measured by a machine, and finally 2 is utilized -△△CT And processing the data. The reference gene is ZmUBQ.
Real-time PCR primers were as follows (5 '-3'):
ZmPAL3-Primer1：GCTGCCCATCAACATCAACT；
ZmPAL3-Primer2：GTGGCACAATCTGGAGACATACA；
ZmHCT-Primer1：GCCTACCTCCTGAACAACCC；
ZmHCT-Primer2：AGTGCTGACAGATCCGGTTG；
Zm4CL1-Primer1：AGGTTTTAGTTTGTCTGTTTGTTGG；
Zm4CL1-Primer2：TAATTTTCAAGGGATTTTTCTCTCC；
ZmUBQ-Primer1：CTGGTGCCCTCTCCATATGG；
ZmUBQ-Primer2：CAACACTGACACGACTCATGACA。
further test results indicate that lignin synthesis genes in MDR1 overexpressing material (MDR 1OE-1 and MDR1OE-2 of C in fig. 3) are generally up-regulated in expression compared to control ND101, wherein the up-regulated differences between zpal 3 and ZmHCT reach significant levels (< 0.01). This further verifies that the synthesis of corn stalk lignin can be regulated by regulating MDR1 expression, thereby affecting the stalk strength of the plant, and thus regulating the lodging resistance of the plant.
Example 4 Potassium content detection
T for MDR1 overexpressing line 1 (MDR 1 OE-1) 3 T of the generation plants, line 2 (MDR 1 OE-2) 3 The generation plants and the maize inbred line ND101 are subjected to low-potassium and high-potassium hydroponic treatment under greenhouse conditions, and the third, fourth and fifth leaves obtained from the hydroponic treatment to the V6 stage are subjected to phenotypic observation, potassium content determination and potassium ion distribution observation and analysis.
1. Seedling stage MDR1 over-expression material low-potassium and high-potassium hydroponic phenotype observation
The normal plants have obviously reduced potassium content in the body after the low-potassium treatment, and simultaneously have the phenotype of yellowing leaf edges and reduced green-keeping performance, so that the green-keeping performance of the leaf can be used as an index for reflecting the change of the potassium content in the body of the plant after the low-potassium treatment. As shown in the observation results in the water culture experiment in FIG. 4, under the low-potassium water culture condition (represented by LK in FIG. 4A), the green-keeping property of the leaves of the old leaves of the MDR1 over-expression materials (MDR 1OE-1 and MDR1OE-2 in FIG. 4A) is better than that of the control material ND101; under high potassium hydroponic conditions (represented by HK in fig. 4 a), MDR1 overexpressing material was not significantly different from control. This suggests that MDR1 is involved in regulating leaf potassium ion balance (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, regulatory K+ transporter AKT1 in Arabidopsis).
2. Determination of Potassium content
Drying, crushing and weighing materials:
the corn material obtained (base third leaf) was dried (3-5 days) to constant weight in an 80 ℃ oven, and the dried material was weighed using an analytical balance and recorded as w (g). If the dry weight of the material is greater than 0.2g, it is necessary to crush it. The application adopts a western medicine powder beater to fully crush the materials. After pulverization, about 0.2g of the sample was weighed again and the dry weight was recorded as w (g).
(1) Acid soaking and drying of crucible
Selecting a sufficient amount of 30mL crucible and cover to perform 0.1N HCl (8.68 mL concentrated hydrochloric acid is measured and added to the solution containing ddH) 2 O1L flask was filled with ddH 2 O to volume 1L) was immersed overnight, followed by natural drying or oven drying at 80 ℃.
(2) Carbonization and ashing of materials
And (3) loading the weighed materials into a dried crucible, and placing the crucible in a muffle furnace to carry out carbonization at 300 ℃ for 8 hours and ashing at 575 ℃ for 10 hours. The muffle furnace is closed, and the furnace is carefully taken out when the temperature is reduced to room temperature.
(3) Dissolving and filtering hydrochloric acid
20mL of 0.1N HCl was added to each crucible and dissolved overnight. During the process, the solution can be blown and sucked by a gun head to be uniformly mixed. The next day is filtered by using a 0.22 μm water filter head, and finally 5-10mL of solution is obtained.
(4) Solution dilution and on-machine measurement
The standard curve was prepared using 200mM KCl and 0.1N HCl to prepare solutions with potassium concentrations of 0, 25, 50, 75, 100, 125, 150, 175 and 200. Mu.M in this order, and using an on-machine microwave plasma atomic emission spectrometer. Taking a proper amount of solution for pre-measurement, and diluting if the measurement range is exceeded, wherein the dilution factor is recorded as n. In general, dilution to 100. Mu.M or more is preferable, and the range is not more than. The potassium concentration measurement was noted as x (μm). The potassium content calculation formula: [ K ] + ](mmol/g)＝nx×20/1000/1000/w＝0.00002nx/w。
The results of the potassium content determination of the basal third leaf show that the potassium content of the MDR1 over-expression material (MDR 1OE-1 and MDR1OE-2 of FIG. 4B) under the low-potassium hydroponic condition (LK of FIG. 4B) is significantly higher than that of the control material ND101; whereas under high potassium hydroponic conditions (HK of B in fig. 4), the overexpressing material was not significantly different from the control. The results show that MDR1 is involved in regulating and controlling potassium ion balance of the leaves.
3. Potassium ion distribution observation
High resolution internode potassium ion distribution observation was done using an X-ray fluorescence scanner (M4 Tornado, bruker). Cutting the ear part in the male stage into slices with relatively consistent thickness by hands, adhering the slices to a glass plate of an X-ray fluorescence spectrometer by using a special adhesive tape, and setting parameters as follows according to the instructions of manufacturers: excitation, 50kV, 600. Mu.A; vacuum path, silicon drift detector, detector energy resolution <150eV; the spot size of the X-ray electron beam is less than or equal to 20 mu m. Potassium ion abundance is indicated by fluorescence intensity.
As a result of detecting the distribution and relative content of potassium content in vivo by using an X-ray fluorescence spectrometer, it was found that the fluorescence intensity of the ear stems of the MDR1 overexpressing materials (MDR 1OE-1 and MDR1OE-2 of FIG. 4C) was significantly higher than that of the control material ND101 under the low-potassium condition. The potassium content of the MDR1 over-expression materials (MDR 1OE-1 and MDR1 OE-2) is measured, and the result is consistent with the X-ray scanning result, and the potassium content of the ear stems of the MDR1 over-expression materials (MDR 1OE-1 and MDR1 OE-2) under the low-potassium condition (represented by (D) in FIG. 4) is obviously higher than that of a control ND101 (P < 0.01); under high potassium conditions, the difference between the MDR1 over-expressed material and the control is not obvious, which indicates that MDR1 participates in maintaining potassium balance of tissues near the ear node. In combination with seedling stage results, MDR1 plays an important role in potassium ion distribution process between leaves and internodes.
In conclusion, the MDR1 gene in the corn is involved in regulating and controlling lignin content of corn stalks, potassium ion distribution and balance between corn leaves and internodes, so that lodging resistance of the corn is regulated and controlled. In actual production, the MDR1 gene expression can be regulated and controlled to be applied to lodging-resistant breeding and improvement of corn.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.
Claims (10)
1. Use of a protein and/or a substance that modulates the expression of a gene encoding said protein and/or a substance that modulates the content of said protein and/or a substance that modulates the activity of said protein, for any of the following:
p1, in regulating and controlling or reducing plant lodging rate,
p2, in regulating and controlling or improving the bending force of plant stems,
p3, in regulating and controlling or improving the lignin content of the plant stalks,
p4, in regulating and controlling or promoting the potassium ion balance of plants,
p5, in regulating and controlling or improving the potassium ion content of plant leaves,
p6, application in plant lodging-resistant breeding,
p7, application in plant quality improvement;
the protein is the protein of A1), A2) or A3) as follows:
a1 Amino acid sequence is protein of sequence 1 in a sequence table;
a2 Protein which is obtained by substituting and/or deleting and/or adding amino acid residues on the amino acid sequence shown in the sequence 1 in the sequence table, is derived from A1) and has the same function or has more than 80 percent of identity with the protein shown in A1) and has the same function;
a3 Fusion proteins obtained by ligating protein tags at the N-terminus or/and the C-terminus of A1) or A2).
2. The use according to claim 1, characterized in that: the protein is derived from corn.
3. Use according to claim 1 or 2, characterized in that: the plant is any one of the following:
c1 Dicotyledonous plants;
d1 A monocotyledonous plant,
d2 A plant of the family Gramineae,
d3 A plant of the order Gramineae,
d4 A maize plant;
d5 Corn).
4. Use of any of the following biological materials in relation to the protein as claimed in claim 1 or 2:
q1, the application of the biological material in regulating and controlling or reducing the plant lodging rate,
q2, the application of the biological material in regulating and controlling or improving the bending force of plant stems,
q3, the application of the biological material in regulating and controlling or improving the lignin content of the plant stalks,
q4, the application of the biological material in regulating or promoting the potassium ion balance of plants,
q5, the application of the biological material in regulating and controlling or improving the potassium ion content of plant leaves,
q6, the application of the biological material in plant lodging-resistant breeding,
q7, application of the biological material in plant quality improvement;
the biomaterial is any one of the following B1) to B9):
b1 A nucleic acid molecule encoding the protein of claim 1;
b2 An expression cassette comprising the nucleic acid molecule of B1);
b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);
b4 A recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);
b5 A transgenic plant cell line comprising the nucleic acid molecule of B1) or a transgenic plant cell line comprising the expression cassette of B2);
b6 A transgenic plant tissue comprising the nucleic acid molecule of B1) or a transgenic plant tissue comprising the expression cassette of B2);
b7 A transgenic plant organ comprising the nucleic acid molecule of B1) or a transgenic plant organ comprising the expression cassette of B2);
b8 A nucleic acid molecule which promotes or enhances the gene expression of the protein of claim 1;
b9 An expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing the nucleic acid molecule of B8).
5. The use according to claim 4, characterized in that: b1 The nucleic acid molecule is a gene encoding the protein as shown in b 1), b 2) or b 3) below:
b1 A coding sequence of the coding chain is a cDNA molecule or a DNA molecule of a nucleotide of a sequence 2 in a sequence table;
b2 Nucleotide is a DNA molecule of a sequence 3 in a sequence table,
b3 A cDNA molecule or a DNA molecule which hybridizes with the cDNA or DNA molecule defined in b 2) and which codes for a protein having the same function.
6. Use according to claim 4 or 5, characterized in that: the plant is any one of the following:
c1 Dicotyledonous plants;
d1 A monocotyledonous plant,
d2 A plant of the family Gramineae,
d3 A plant of the order Gramineae,
d4 A maize plant;
d5 Corn).
7. A method for reducing plant lodging rate, comprising enhancing or increasing expression of a gene encoding the protein of claim 1 in a plant of interest or/and regulating the content of the protein or/and activity of the protein of claim 1, thereby reducing plant lodging rate.
8. The method according to claim 7, wherein: the enhancement or improvement of the activity of the protein of claim 1 or/and the expression level of the gene encoding the protein of claim 1 in a plant of interest is achieved by introducing the gene encoding the protein of claim 1 into the plant of interest.
9. The method according to claim 7 or 8, characterized in that: the plant and/or the target plant is any one of the following:
c1 Dicotyledonous plants;
d1 A monocotyledonous plant,
d2 A plant of the family Gramineae,
d3 A plant of the order Gramineae,
d4 A maize plant;
d5 Corn).
10. The protein of claim 1 and/or the biomaterial of claim 4 or 5.
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