CN114644699B - Application of Substances Regulating ZmARP1 Gene Expression in Regulating Plant Drought Resistance - Google Patents

Abstract

The invention discloses the application of a substance regulating ZmARP1 gene expression in regulating plant drought resistance. A technical solution to be protected by the present invention is the application of substances regulating gene expression in regulating plant drought resistance. The protein encoded by the gene is ZmARP1 protein, and its amino acid sequence is the protein of sequence 1 in the sequence list or the sequence in the sequence list A protein derived from the protein of 1 having more than 80% identity and the same function. In the present invention, the ZmARP1 gene from maize 1145 is introduced into maize B73 to obtain a transgenic overexpression line of ZmARP1; compared with the untransformed B73 control plant, the overexpression of the ZmARP1 gene increases the sensitivity of the transgenic maize to drought stress. It shows that ZmARP1 is involved in the regulation and adaptation of plants to drought-related stress.

Description

Application of Substances Regulating ZmARP1 Gene Expression in Regulating Plant Drought Resistance

technical field

The invention relates to the field of biotechnology, in particular to the application of substances regulating ZmARP1 gene expression in regulating plant drought resistance.

Background technique

Corn is one of the three major food crops in the world. It is planted in a wide range of areas, but the distribution in each area is not uniform. Different areas have different environments, different climatic conditions, and different precipitation. Drought is an important factor affecting maize yield. It will inhibit the growth rate of maize at the seedling stage, resulting in a severe shortening of the development period; it will also inhibit the plant height of maize, resulting in wilting of leaves, thereby reducing photosynthesis. During the grain-fill stage of corn, drought can lead to underfilled kernels, which can lead to reduced corn yields.

ARP1 is a protein regulated by auxin and has an unknown functional domain. There have been some studies on ARP1 protein in plants. The homologous protein in Arabidopsis is SOSEKI. Previous reports have shown that the homologous protein of Arabidopsis can participate in The polarity of cell growth and can affect the direction of cell division. There are few related studies on ZmARP1 in maize.

Contents of the invention

The technical problem to be solved by the present invention is to determine the function and application of the ZmARP1 protein and/or how to regulate the drought resistance of plants.

In order to solve the problems of the technologies described above, the present invention firstly provides any of the following applications of substances regulating the expression of the ZmARP1 gene in corn:

F1. The application of substances that regulate gene expression in regulating plant drought resistance,

F2. The application of substances that regulate gene expression in the preparation of products that reduce plant drought resistance,

F3. Application of substances regulating gene expression in plant breeding;

In F1-F3, the gene encodes a protein (named ZmARP1) as follows A1), A2) or A3):

A1) The amino acid sequence is the protein of sequence 1 in the sequence listing;

A2) The amino acid sequence shown in Sequence 1 in the sequence listing is obtained by substitution and/or deletion and/or addition of one or several amino acid residues and has the same function derived from A1) or shown in A1) Proteins with more than 80% identity and the same function;

A3) A fusion protein obtained by linking protein tags at the N-terminus or/and C-terminus of A1) or A2).

The aforementioned plant may be maize.

Among the above proteins, sequence 1 in the sequence listing consists of 421 amino acid residues.

The above-mentioned proteins can be synthesized artificially, or their coding genes can be synthesized first, and then biologically expressed.

Among the above proteins, the protein-tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology, so as to facilitate the expression, detection, tracking and/or purification of the target protein. The protein tag can be Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag and/or SUMO tag, etc.

In the above-mentioned proteins, the identity refers to the identity of amino acid sequences. Amino acid sequence identities can be determined using homology search sites on the Internet, such as the BLAST webpage of the NCBI homepage. For example, in advanced BLAST2.1, by using blastp as the program, set the Expect value to 10, set all Filters to OFF, use BLOSUM62 as Matrix, and set Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (the default value) and search for the identity of a pair of amino acid sequences to calculate, and then the value (%) of the identity can be obtained.

Among the above proteins, the above 80% identity can be at least 81%, 82%, 85%, 86%, 88%, 90%, 91%, 92%, 95%, 96%, 98%, 99% Or 100% identity.

In the above application, the substance that regulates gene expression can be a substance that performs at least one regulation in the following six kinds of regulation: 1) regulation at the gene transcription level; 2) regulation after the gene transcription (that is, the regulation of the splicing or processing of the primary transcript of the gene); 3) the regulation of the RNA translocation of the gene (that is, the regulation of the mRNA of the gene from the nucleus to the cytoplasm) 4) regulation of the translation of the gene; 5) regulation of the degradation of the mRNA of the gene; 6) regulation of the post-translation of the gene (that is, the activity of the protein translated by the gene regulation).

In the above application, the regulating the drought resistance of plants is to increase the sensitivity of plants to drought.

In the above application, the regulating gene expression can be to enhance or improve the gene expression.

In the above application, the substance regulating gene expression may be a reagent that enhances or improves the gene expression.

The substance that regulates gene expression can specifically be any of the following:

B1) a nucleic acid molecule encoding the above-mentioned protein;

B2) an expression cassette containing the nucleic acid molecule of B1);

B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2).

The above-mentioned proteins also belong to the protection scope of the present invention.

In order to solve the above-mentioned technical problems, the present invention also provides any of the following applications of the above-mentioned proteins:

P1. The application of the above-mentioned proteins in regulating the drought resistance of plants,

P2, the application of the above-mentioned protein in the preparation of products that reduce the drought resistance of plants,

P3. Application of the above protein in plant breeding.

In order to solve the above-mentioned technical problems, the present invention also provides any of the following applications of the above-mentioned protein-related biological materials:

Q1. The application of the biological material in regulating and controlling the drought resistance of plants,

Q2. The application of the biological material in the preparation of products that reduce the drought resistance of plants,

Q3. The application of the biological material in plant breeding.

The biological material is any one of the following B1) to B11):

B1) a nucleic acid molecule encoding the above-mentioned protein;

B2) an expression cassette containing the nucleic acid molecule of 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 a 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) a transgenic plant tissue containing the nucleic acid molecule described in B1), or a transgenic plant tissue containing the expression cassette described in B2);

B7) a transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2);

B8) Nucleic acid molecules that promote or increase the gene expression of the above-mentioned proteins;

B9) expression cassettes, recombinant vectors, recombinant microorganisms or transgenic plant cell lines containing the nucleic acid molecules described in B8);

B10) Nucleic acid molecules that inhibit or reduce the gene expression of the above-mentioned proteins;

B11) An expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing the nucleic acid molecule of B10).

In the above biological material, the nucleic acid molecule in B1) is the gene encoding the protein shown in b1), b2) or b3) as follows:

b1) The coding sequence of the coding strand is a cDNA molecule or a DNA molecule of the nucleotide sequence 3 in the sequence listing;

b2) The nucleotide is a cDNA molecule or a DNA molecule of sequence 3 in the sequence listing;

b3) A cDNA molecule or a DNA molecule that hybridizes to the cDNA or DNA molecule defined in b2) and encodes a protein having the same function.

Among the above-mentioned biological materials, the expression cassette containing nucleic acid molecules described in B2) refers to the DNA capable of expressing the protein described in the above-mentioned application in the host cell. A terminator that terminates transcription of a gene encoding a protein is included. 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. An existing plant expression vector can be used to construct a recombinant expression vector containing the expression cassette of the protein-coding gene. The plant expression vectors include binary Agrobacterium vectors and vectors that 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 Company), etc.

Among the above biological materials, the recombinant microorganisms can specifically be yeast, bacteria, algae and fungi.

Any of the following products of the above-mentioned substances regulating genes and/or the above-mentioned proteins and/or the above-mentioned biological materials also belong to the protection scope of the present invention:

D1. Products that regulate the drought resistance of plants;

D2. Products that reduce the drought resistance of plants;

D3. Products that increase the drought sensitivity of plants.

In the above, the product that reduces the drought resistance of plants can be a plant stress regulator. The plant stress regulator may contain the above-mentioned protein or/and the biological material. In order to solve the above technical problems, the present invention also provides a method for cultivating drought-sensitive plants, including increasing the biological expression of the above-mentioned protein or/and the expression of the above-mentioned nucleic acid molecule in the target plant to obtain drought-sensitive plants. The drought-sensitive plant is more sensitive to drought than the plant of interest. The target plant can be maize.

In the above method, the increase of the ZmARP1 protein content or/and the expression level of the above nucleic acid molecules in the target plant can be achieved by introducing the ZmARP1 protein encoding gene into the target plant.

In the above method, the stress-sensitive plants may be transgenic plants, or plants obtained by conventional breeding techniques such as hybridization.

In the above method, the transgenic plant is understood to include not only the first to second generation transgenic plants, but also their progeny. For transgenic plants, the gene can be propagated in that species, or transferred into other varieties of the same species, particularly including commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, callus, whole plants and cells.

Modulating the drought resistance of plants as described above is to increase the sensitivity of plants to drought.

The plant described above may be maize.

The above-mentioned nucleic acid molecules can produce different transcripts and translate different proteins, and the different transcripts produced by the nucleic acid molecules and the different proteins translated are all within the protection scope of this patent.

In the present invention, the ZmARP1 gene from maize 1145 is introduced into maize B73 to obtain a transgenic overexpression line of ZmARP1; compared with the untransformed B73 control plant, the overexpression of the ZmARP1 gene increases the sensitivity of the transgenic maize to drought stress. It shows that ZmARP1 is involved in the regulation and adaptation of plants to drought-related stress.

Description of drawings

Figure 1 shows the drought-treated phenotypes of the control and ZmARP1 overexpression lines.

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guideline for those skilled in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The Agrobacterium strain was EHA105. The main reagents include: restriction endonuclease, DNA polymerase, T4 ligase, etc. from biological companies such as NEB and Toyobo; reverse transcription kit from Thermo Company; RNA extraction kit from Magen Company; quantitative PCR reagent from Taraka Company; Plasmid extraction kits and DNA recovery kits were purchased from Tiangen Company; MS medium, agar powder, agarose, ampicillin, kanamycin, gentamicin sulfate, rifampin and other antibiotics were purchased from sigma; Various other chemical reagents used in the examples were imported or domestic analytical reagents; primer synthesis and sequencing were completed by Yingjun Company.

Construction of embodiment 1 ZmARP1 gene overexpression vector

The inventors of the present application discovered a gene related to plant drought tolerance in maize ecotype 1145. The nucleotide sequence of the genome gene is shown in sequence 2 in the sequence listing, and the gene consists of 2879 bases. The reading frame of the gene T01 transcript is from the 1st to the 2501st base at the 5' end. The transcript has 3 exons, of which there are 2 coding exons, which are the 1st to 971st bases in the reading frame and the 2207th to 2501st bases in the reading frame, and the rest are included subsequence.

The protein encoded by this gene is named ZmARP1, its amino acid sequence is shown in sequence 1 in the sequence listing, and the CDS of the cDNA gene of ZmARP1 is shown in sequence 3 in the sequence listing.

Insert the DNA molecule whose nucleotide sequence is sequence 3 in the sequence table into the pBCXUN vector to obtain the ZmARP1 gene expression recombinant vector, which is named pBCXUN-ZmARP1. In pBCXUN-ZmARP1, the DNA molecule shown in sequence 3 in the sequence listing is initiated by the promoter of the maize ubiquitin gene Ubi and terminated by the Nos terminator, capable of expressing ZmARP1 protein (the amino acid sequence is shown in sequence 1 in the sequence listing).

The pBCXUN vector replaces the HYG gene (hptII, hygromycin resistance gene) of pCXUN (GenBank: FJ905215.1, 06-JUL-2009) with the Bar gene (encoding phosphinothricin acetyltransferase) (GenBank: MG719235. The 284th-835th nucleotide in 1, 02-OCT-2018), the expression vector obtained by keeping the other nucleotides of pCXUN unchanged.

The acquisition of embodiment 2ZmARP1 gene overexpression plant

The pBCXUN-ZmARP1 constructed in Example 1 was transformed into the competent Agrobacterium EHA105 strain by heat shock method, positive clones were identified by colony PCR, positive clones were selected for sequencing, colony PCR and sequencing identified primers as UbiP-seq (corresponding to UbiP-seq) in the terminator) and NosR-seq (corresponding to the Nos terminator). The positive clone containing pBCXUN-ZmARP1 is the recombinant Agrobacterium, named pBCXUN-ZmARP1/EHA105.

UbiP-seq: 5′-TTTTAGCCCTGCCTTCATACGC-3′,

NosR-seq: 5′-AGACCGGCAACAGGATTCAATC-3′.

Inoculate a single colony of pBCXUN-ZmARP1/EHA105 into 2-3 mL of liquid medium containing 100 μg/mL kanamycin and 50 μg/mL rifampicin, culture with shaking at 28°C overnight, and transfer to a culture medium containing kanamycin the next day. Shake culture in a large amount of liquid medium containing antibiotics and rifampicin antibiotics, collect the bacteria after several transfers, and resuspend the bacteria until the OD ₆₀₀ is between 0.8-1.0 to obtain the recombinant Agrobacterium suspension. After the obtained recombinant Agrobacterium suspension was used to infect the B73 maize immature embryos picked out under aseptic conditions, the callus was induced to form seedlings by screening with the herbicide glufosinate, and the ZmARP1 transgenic plants (extracted plants) were screened by PCR identification method. The genomic DNA of the leaf was amplified by PCR using a primer pair composed of UbiP-seq and NosR-seq, and the plants that obtained specific amplification products were ZmARP1 transgenic plants). ZmARP1 transgenic plants were selfed and multiplied to obtain T3 generation stable ZmARP1 gene overexpressed plants for subsequent experiments.

Example 3 Detection of ZmARP1 Gene Overexpression Maize Drought Treatment Phenotype

Experiments consisted of three replicates. Each replicate contained 15 small pots of control (B73) and 15 small pots of ZmARP1 gene overexpressed plants (T3 generation). Add 140g of soil to each small pot, add water to the tray, put 4 seeds in each small pot, cover with 50ml of soil, pour out the remaining water in the tray after absorbing the water, and put it in the cultivation room (temperature 25°C, humidity 40°C) %) normal cultivation, remove a seedling with uneven growth after emergence, add 1L of water in the tray, drain the water after it is full, and start the drought treatment and no longer water. Drought stress phenotypes appeared after 3 weeks of treatment, and the results were shown in Figure 1. ZmARP1 transgenic overexpression plants (shown by OE ZmARP1 in Figure 1) grew worse than the control B73 (shown by WT in Figure 1). Plants of the two materials showed wilting phenotypes of leaves under drought stress, and the wilting degree of OE ZmARP1 plants was higher than that of WT plants, indicating that ZmARP1 transgenic plants were more sensitive to drought than control B73 plants, and ZmARP1 protein played a negative role in maize's resistance to drought adversity stress effect.

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experiments, the present invention can be practiced in a wider range under equivalent parameters, concentrations and conditions. While specific embodiments of the invention have been shown, it should be understood that the invention can be further modified. In a word, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by using conventional techniques known in the art and departing from the disclosed scope of this application. Applications of some of the essential features are possible within the scope of the appended claims below.

sequence listing

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<120> Application of Substances Regulating ZmARP1 Gene Expression in Regulating Plant Drought Resistance

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agttaa 1266

Claims (7)

1. The application of protein in cultivating plants with reduced drought resistance; The protein is the following A1) or A2) protein: A1) The amino acid sequence is the protein of sequence 1 in the sequence listing; A2) A fusion protein obtained by linking protein tags at the N-terminus or/and C-terminus of A1); The plant is a monocot. 2. The application according to claim 1, characterized in that: the plant is corn. 3. The application of biological material related to the protein described in claim 1 or 2 in cultivating plants with reduced drought resistance; The biological material is any of the following: B1) a nucleic acid molecule encoding the protein of claim 1; B2) an expression cassette containing the nucleic acid molecule of 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 an expression cassette described in B2), or a recombinant microorganism containing a recombinant vector described in B3); The plant is a monocot. 4. The application according to claim 3, characterized in that: B1) the nucleotide sequence of the nucleic acid molecule is as shown in sequence 3 in the sequence listing. 5. The application according to claim 3 or 4, characterized in that: the plant is corn. 6. A method for cultivating drought-sensitive plants, comprising improving the biological expression of the protein described in claim 1 or 2 in the target plant or/and the expression of the nucleic acid molecule described in claim 3 or 4, to obtain drought-sensitive A plant; the drought-sensitive plant is more sensitive to drought than the target plant; the plant is a monocot. 7. The method of claim 6, wherein the plant is maize.

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