CN116676331A - Application of ZmST1 protein and coding gene thereof in regulation and control of green-keeping, disease resistance and yield of plants - Google Patents
Application of ZmST1 protein and coding gene thereof in regulation and control of green-keeping, disease resistance and yield of plants Download PDFInfo
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- CN116676331A CN116676331A CN202310784939.0A CN202310784939A CN116676331A CN 116676331 A CN116676331 A CN 116676331A CN 202310784939 A CN202310784939 A CN 202310784939A CN 116676331 A CN116676331 A CN 116676331A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
- C12N15/8269—Photosynthesis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Abstract
The application discloses application of ZmST1 protein and a coding gene thereof in regulating and controlling green-keeping, disease resistance and yield of plants. The application constructs a ZmST1 over-expression strain by over-expressing ZmST1 protein in corn, and discovers by analyzing the phenotype and yield of the ZmST1 over-expression strain of green-keeping and disease-resistant leaves in the mature period: compared with the wild type, the ZmST1 over-expression strain shows visible functional green-keeping prolongation, field disease resistance is enhanced, and yield is increased, which indicates that the ZmST1 protein or the coding gene thereof can regulate and control the green-keeping and disease resistance of the leaves in the mature period of corn by participating in the transportation of photosynthesis products in corn chloroplasts, delay the aging process of the leaves, prolong the green-keeping functional period of the plant leaves and improve the yield of corn. The application has important significance for cultivating high-yield corns.
Description
Technical Field
The application belongs to the technical field of biological genes, and particularly relates to ZmST1 protein and application of a coding gene thereof in regulating and controlling plant green-keeping property, disease resistance and yield.
Background
Corn is one of three main grain crops, and is the crop with the widest planting area and highest yield at present; in addition, corn is also an important feed crop and industrial feedstock. Therefore, improving the corn yield plays a significant role in guaranteeing the grain safety and meeting the market demand. Currently, the key to improving crop yield mainly comprises three aspects: 1. improving photosynthesis ability of 'source' tissue; 2. improving the unloading capacity of phloem sugar; 3. improving the unloading and storage capacity of sugar in the "pool" organization. The plant is used as autotrophic organism, and light energy is converted into chemical energy through photosynthesis of chloroplast, and the chemical energy exists mainly in the form of sugar, so that energy is provided for plant growth and development. The transport of sugar from the "source" tissue to the "sink" tissue affects the various stages of vegetative and reproductive growth of the plant, particularly the redistribution of photosynthesis products and the ability to transport to the "sink" tissue directly affects the size of the "sink" organ and the crop yield. Whereas sugar transporters play a key role in the transport of sugar from "source" tissues to "sink" tissues in plants.
ATP binding cassette transporters are used as one of the largest protein subfamily groups in plants, and transport substrates include substances such as saccharides, metal ions, auxins and the like, and participate in a plurality of physiological and biochemical processes. There are 133 ATP-binding cassette transporter members in maize, but there is little research on the function of ATP-binding cassette transporters in maize, particularly in the transport of photosynthesis products and the partitioning of sugars, and further investigation is still needed.
Disclosure of Invention
It is an object of the present application to provide novel uses of ZmST1 protein or biological materials related to ZmST1 protein.
The present application provides the use of ZmST1 protein or a biomaterial associated with ZmST1 protein in any one of the following 1) -5):
1) Regulating and controlling the green keeping performance of plants;
2) Regulating and controlling the plant yield;
3) Regulating and controlling plant disease resistance;
4) Cultivating a transgenic plant with increased stay green and/or increased yield and/or increased disease resistance;
5) Plant breeding;
the ZmST1 protein is a 1) or a 2) or a 3) or a 4):
a1 Amino acid sequence is a protein shown in sequence 3;
a2 A fusion protein obtained by ligating a tag to the N-terminus or/and the C-terminus of the protein represented by the sequence 3;
a3 Protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 3 and is related to green, disease resistance or yield of plants;
a4 90% identical to the amino acid sequence shown in sequence 3, derived from maize and associated with green, disease resistance or yield in plants.
Wherein, sequence 3 consists of 220 amino acid residues.
The protein of a 2), wherein the tag refers to a polypeptide or protein which is fused and expressed together with the target protein by using a DNA in vitro recombination technology, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc.
The protein according to a 3) above, wherein the substitution and/or deletion and/or addition of one or several amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues or a substitution and/or deletion and/or addition of not more than 9 amino acid residues or a substitution and/or deletion and/or addition of not more than 8 amino acid residues or a substitution and/or deletion and/or addition of not more than 7 amino acid residues or a substitution and/or deletion and/or addition of not more than 6 amino acid residues or a substitution and/or deletion and/or addition of not more than 5 amino acid residues or a substitution and/or deletion and/or addition of not more than 4 amino acid residues or a substitution and/or deletion and/or addition of not more than 3 amino acid residues or a substitution and/or deletion and/or addition of not more than 2 amino acid residues or a substitution and/or deletion and/or addition of not more than 1 amino acid residue.
The protein according to a 4) above, wherein the identity is the identity of an amino acid sequence. 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. Such identity includes amino acid sequences having 90% or more, or 91% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more homology to the amino acid sequences shown in sequence 3 of the present application.
The protein described in the above a 1), a 2), a 3) or a 4) can be synthesized artificially or can be obtained by synthesizing the coding gene and then biologically expressing.
The biomaterial is any one of the following A1) to A8):
a1 Nucleic acid molecules encoding ZmST1 proteins;
a2 An expression cassette comprising A1) said nucleic acid molecule;
a3 A) a recombinant vector comprising the nucleic acid molecule of A1);
a4 A recombinant vector comprising the expression cassette of A2);
a5 A) a recombinant microorganism comprising the nucleic acid molecule of A1);
a6 A) a recombinant microorganism comprising the expression cassette of A2);
a7 A) a recombinant microorganism comprising the recombinant vector of A3);
a8 A recombinant microorganism comprising the recombinant vector of A4).
Further, the nucleic acid molecule of A1) is a gene represented by the following B1) or B2) or B3) or B4):
b1 A DNA molecule represented by sequence 1;
b2 A DNA molecule represented by SEQ ID No. 2;
b3 A DNA molecule which has 75% or more identity to the nucleotide sequence defined in B1) or B2) and which encodes the ZmST1 protein described above;
b4 A DNA molecule which hybridizes under stringent conditions to the nucleotide sequence defined in B1) or B2) or B3) and which codes for the ZmST1 protein described above.
Wherein 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, etc.
The nucleotide sequence encoding the ZmST1 protein of the present application can be easily mutated by those skilled in the art using known methods such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence encoding the above ZmST1 protein are derived from the nucleotide sequence of the present application and are equivalent to the sequence of the present application as long as the above ZmST1 protein is encoded and has the same function.
The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 75% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of a protein consisting of the amino acid sequence shown in the coding sequence 3 of the present application. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.
The 75% or more identity may be 80%, 85%, 90% or 95% or more identity.
In the above application, the stringent conditions are hybridization and washing the membrane 2 times at 68℃in a solution of 2 XSSC, 0.1% SDS for 5min each time, and hybridization and washing the membrane 2 times at 68℃in a solution of 0.5 XSSC, 0.1% SDS for 15min each time; alternatively, hybridization and washing of the membrane were performed at 65℃in a solution of 0.1 XSSPE (or 0.1 XSSC) and 0.1% SDS.
In the above applications, the expression cassette containing a nucleic acid molecule encoding a ZmST1 protein (ZmST 1 gene expression cassette) described in A2) refers to a DNA capable of expressing a ZmST1 protein in a host cell, which DNA may include not only a promoter for initiating transcription of ZmST1 but also a terminator for terminating transcription of ZmST 1. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present application include, but are not limited to: a constitutive promoter; 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 terminator.
Recombinant vectors containing the ZmST1 gene expression cassette can be constructed using existing 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, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb, etc. 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. The sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene. To facilitate identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, for example by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), antibiotic marker genes (such as nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to the herbicide phosphinothricin, hph gene conferring resistance to antibiotic hygromycin, dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate) or chemical marker genes, etc. (such as herbicide resistance genes), mannose-6-phosphate isomerase gene providing mannose metabolization ability, etc. From the safety of transgenic plants, transformed plants can be screened directly in stress without adding any selectable marker gene.
In the above applications, the vector may be a plasmid, cosmid, phage or viral vector.
In the above application, the microorganism may be a yeast, a bacterium, an alga or a fungus, such as Agrobacterium.
In the above application, the regulation of plant green-keeping performance is to improve plant green-keeping performance, in particular to improve plant leaf green-keeping performance, such as improving plant mature stage leaf green-keeping performance.
The regulation of plant yield is to increase plant yield, in particular to increase plant seed weight.
The regulation of plant disease resistance is to improve plant disease resistance, and in particular to improve the resistance of plants to spike and grain rot.
The object of said plant breeding is to cultivate plant varieties with high stay green and/or high yield and/or high disease resistance.
It is a final object of the present application to provide a method for breeding transgenic plants with increased stay green and/or increased yield and/or increased disease resistance.
The method for cultivating the transgenic plant with improved green-keeping property and/or improved yield and/or improved disease resistance comprises the following steps: increasing the content and/or activity of ZmST1 protein in the recipient plant to obtain a transgenic plant; the transgenic plants have a green-keeping, yield and/or disease resistance higher than the recipient plants.
Further, the method of increasing the amount and/or activity of a ZmST1 protein in a recipient plant is to overexpress the ZmST1 protein in the recipient plant.
The over-expression method is to introduce the coding gene of ZmST1 protein into a receptor plant.
Furthermore, the coding gene sequence of the ZmST1 protein is shown as a sequence 2 in a sequence table.
In any of the above applications or methods, the transgenic plant is understood to include not only the first generation transgenic plant obtained by transforming the ZmST1 gene into a recipient plant, but also its progeny. For transgenic plants, the gene may be propagated in that species, and may be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, calli, whole plants and cells.
In any of the above applications or methods, the plant is a dicotyledonous plant or a monocotyledonous plant. Further, the monocot plant may be maize. Still further, the variety of corn is maize inbred line ND101.
The application constructs a ZmST1 over-expression strain by over-expressing ZmST1 protein in corn, and discovers by analyzing the phenotype and yield of the ZmST1 over-expression strain of green-keeping and disease-resistant leaves in the mature period: compared with the wild type, the ZmST1 over-expression strain shows visible functional green-keeping prolongation, field disease resistance is enhanced, seeds become larger, and yield is increased, which indicates that the ZmST1 protein or the coding gene thereof can regulate and control the green-keeping and disease resistance of the leaves in the mature period of corn by participating in the transportation of photosynthesis products in corn chloroplasts, delay the aging process of the leaves, prolong the green-keeping functional period of the plant leaves and improve the yield of corn. The application has important significance for cultivating high-yield corns.
Drawings
FIG. 1 is an identification of maize ZmST1 over-expression lines.
FIG. 2 is a phenotype of leaf green retention and disease resistance in mature stage of maize wild type ND101 and ZmST1 overexpressing lines.
FIG. 3 is the yield of maize wild-type ND101 and ZmST1 overexpressing lines.
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. In the following examples, three or more repeated quantitative tests were carried out unless otherwise specified.
Maize inbred line ND101 in the examples below is provided by the China agricultural university crop functional genome and molecular breeding research center and is described in the literature "Liu F, cheng J, liu X, wang X (2021) High-throughput and accurate determination of transgene copy number and zygosity in transgenic maize:from DNAextration dataanalysis.IntJMolSci22: 12487".
The agrobacterium tumefaciens strain EHA105 in the following examples is provided by the chinese agricultural university crop functional genome and molecular breeding research center and is described in document "Chen, s; songakumarn, p.; liu, j.; wang, G. -L.AVersatile Zero Background T-Vector System for Gene Cloning and Functional genomics.plant Physiol.2009,150,1111-1121.
The overexpression vector pCAMBAIA3300-myc in The examples described below is provided by The functional genome and molecular breeding research center of crops at China agricultural university and is described in The literature "Boxin Liu and others, manipulating ZmEXPA4expression ameliorates The drought-induced prolonged anthesis and silking interval in maize, the Plant Cell, volume 33,Issue 6,June 2021,Pages 2058-2071, https:// doi.org/10.1093/pl Cell/koab083.
Example 1 use of ZmST1 protein for improving plant Green-keeping, disease resistance and yield
1. Acquisition and identification of ZmST1 overexpressing lines
1. Obtaining ZmST1 over-expression lines
1) Construction of pCAMBAIA3300-myc-ZmST1 overexpression vector
And (3) replacing the DNA molecule shown in the sequence 2 with the DNA fragment between two XcmI enzyme cutting sites in the overexpression vector pCAMBAIA3300-myc to obtain the pCAMBAIA3300-myc-ZmST1 overexpression vector.
2) Preparation of recombinant bacteria
The pCAMBAIA3300-myc-ZmST1 over-expression vector is introduced into EHA105 agrobacterium, and the EHA105 agrobacterium containing the pCAMBAIA3300-myc-ZmST1 over-expression vector is obtained through identification.
3) Acquisition of transgenic plants
Genetic transformation of young embryo of wild maize inbred line ND101 with EHA105 Agrobacterium containing pCAMBAIA3300-myc-ZmST1 overexpression vector by Agrobacterium-mediated genetic transformation method to obtain T 0 Transgenic maize plants.
4) PCR identification of ZmST1 over-expressed lines
Extraction of T 0 Genomic DNA of the transgenic maize plant is identified by PCR by Bar-F and Bar-R primers, and amplified to form T with 552bp 0 The transgenic corn plants are positive plants, and the homozygous lines are obtained through 2-3 generations of propagation. The primer sequences were as follows:
Bar-F：ATGAGCCCAGAACGACGC；
Bar-R：TCAAATCTCGGTGACGGG。
2. RT-PCR identification of ZmST1 overexpressing lines
Extraction of wild maize inbred ND101 and T 3 The RNA of the generation ZmST1 over-expression homozygous strain is utilized to obtain cDNA by using a TaRaRa reverse transcription kit, the cDNA is used as a template, a specific primer is used for RT-PCR detection, and a Ubiquitin gene is used as an internal reference. The primer sequences were as follows:
UbiP-seq：TTTTAGCCCTGCCTTCATACGC；
NosR-seq：AGACCGGCAACAGGATTCAATC；
Ubiquitin-F：TGGTTGTGGCTTCGTTGGTT；
Ubiquitin-R：GCTGCAGAAGAGTTTTGGGTACA。
the results are shown in FIG. 1, which shows: the wild maize inbred line ND101 and the strain without over-expressing ZmST1 have no bands, and seven ZmST1 over-expressing homozygous lines with different over-expression levels have bands which are respectively marked as ZmST1 over-expressing homozygous lines OE -1 to ZmST1 over-expressing homozygous lines OE -7. Selection of ZmST1 over-expression homozygous lines OE -1 and ZmST1 overexpressing homozygous lines OE -2 was used in the analytical experiments described below.
2. Application of ZmST1 protein in improving green keeping and disease resistance of plants
Test material: t (T) 3 Generation ZmST1 over-expression homozygous line OE -1、T 3 Generation ZmST1 over-expression homozygous line OE -2 and wild maize inbred ND101.
Respectively T 3 Generation ZmST1 over-expression homozygous line OE -1、T 3 Generation ZmST1 over-expression homozygous line OE Seeds of the 2 and wild maize inbred line ND101 (control) were planted in the Hainan-Binnan reproduction base of the university of agriculture, china, in double row areas with a row length of 2.5 meters, a row spacing of 60 cm, a plant spacing of 25 cm, and the phenotype of the plants was observed after the maturity period.
The results are shown in FIG. 2, from which it can be seen that: over-expression of homozygous lines ZmST1 at maturity compared to wild maize inbred line ND101 OE -1 and ZmST1 overexpressing homozygous lines OE The leaves of-2 are not obviously green-losing, and have higher green-keeping performance, and researches find that the green-keeping performance of plants is closely related to stress resistance, especially disease resistance (Sanchez AC, subudhi PK, rosenow DT, nguyen HT.mapping QTLs associated with drought resistance in Sorghum (Sorgum biocolor L. Moench.) Plant Mol biol.2002Mar-Apr;48 (5-6): 713-26.Doi:10.1023/a:1014894130270.PMID: 11999845.). The ZmST1 protein can regulate and control the green-keeping and disease-resistant properties of plants.
3. Application of ZmST1 protein in improving corn yield
Test material: t (T) 3 Generation ZmST1 over-expression homozygous line OE -1、T 3 Generation ZmST1 over-expression homozygous line OE -2 and wild maize inbred ND101.
Respectively T 3 Generation ZmST1 over-expression homozygous line OE -1、T 3 Generation ZmST1 over-expression homozygous line OE 2 and wild maize inbred line ND101 (control) seeds were planted in the Jilin princess mountain experimental base of China university, in double row area with row length of 2.5 meters, row spacing of 60 cm, plant spacing of 25 cm, 3 repetitions, and after maturity, the maize grain weight in each cell was measured, the maize yield measured in this study was the standard cell grain weight, and the calculation formula was: standard cell grain weight = cell grain weight x (1-grain average moisture content)/(1-14%), wherein the grain average moisture content is the average moisture content of seeds obtained by measuring the test material using a finland Wile65 type seed moisture meter.
The results are shown in FIG. 3, from which it can be seen that: the standard cell grain weight of ZmST1 over-expressed homozygous lines was significantly higher than that of the control. Wherein, the average grain weight of a standard cell of the wild corn inbred line ND101 is 0.58 kg, and the ZmST1 overexpresses the homozygous line OE Standard cell particle weight of-1 averages 0.79 kg, zmST1 overexpressing homozygous line OE The standard cell particle weight of-2 averages 0.72 kg. It is shown that ZmST1 protein regulates plant yield.
The above results indicate that ZmST1 protein plays a positive regulatory role in plant green keeping, disease resistance and yield.
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. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (10)
- Use of zmst1 protein in the following 1) -5):1) Regulating and controlling the green keeping performance of plants;2) Regulating and controlling the plant yield;3) Regulating and controlling plant disease resistance;4) Cultivating a transgenic plant with increased stay green and/or increased yield and/or increased disease resistance;5) Plant breeding;the ZmST1 protein is a 1) or a 2) or a 3) or a 4):a1 Amino acid sequence is a protein shown in sequence 3;a2 A fusion protein obtained by ligating a tag to the N-terminus or/and the C-terminus of the protein represented by the sequence 3;a3 Protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 3 and is related to green, disease resistance or yield of plants;a4 90% identical to the amino acid sequence shown in sequence 3, derived from maize and associated with green, disease resistance or yield in plants.
- 2. Use of a biological material related to ZmST1 protein in the following 1) -5):1) Regulating and controlling the green keeping performance of plants;2) Regulating and controlling the plant yield;3) Regulating and controlling plant disease resistance;4) Cultivating a transgenic plant with increased stay green and/or increased yield and/or increased disease resistance;5) Plant breeding;the biomaterial is any one of the following A1) to A8):a1 Nucleic acid molecules encoding ZmST1 proteins;a2 An expression cassette comprising A1) said nucleic acid molecule;a3 A) a recombinant vector comprising the nucleic acid molecule of A1);a4 A recombinant vector comprising the expression cassette of A2);a5 A) a recombinant microorganism comprising the nucleic acid molecule of A1);a6 A) a recombinant microorganism comprising the expression cassette of A2);a7 A) a recombinant microorganism comprising the recombinant vector of A3);a8 A recombinant microorganism comprising the recombinant vector of A4).
- 3. The use according to claim 2, characterized in that: a1 The nucleic acid molecule is a gene represented by the following B1) or B2) or B3) or B4):b1 A DNA molecule represented by sequence 1;b2 A DNA molecule represented by SEQ ID No. 2;b3 A DNA molecule which has 75% or more identity to the nucleotide sequence defined in B1) or B2) and which encodes the ZmST1 protein according to claim 1;b4 A DNA molecule which hybridizes under stringent conditions to the nucleotide sequence defined in B1) or B2) or B3) and which codes for the ZmST1 protein according to claim 1.
- 4. A use according to any one of claims 1-3, characterized in that: the green keeping performance of the control plant is that of the control plant leaf.
- 5. Use according to any one of claims 1-4, characterized in that: the plant is a dicotyledon or monocotyledon; and/or, the monocot is maize.
- 6. A method of growing a transgenic plant with increased stay green and/or increased yield and/or increased disease resistance comprising the steps of: increasing the content and/or activity of the ZmST1 protein of claim 1 in a recipient plant to obtain a transgenic plant; the transgenic plants have a green-keeping, yield and/or disease resistance higher than the recipient plants.
- 7. The method according to claim 6, wherein: the method for increasing the content and/or activity of the ZmST1 protein of claim 1 in a recipient plant is to overexpress the ZmST1 protein in the recipient plant.
- 8. The method according to claim 7, wherein: the over-expression method is to introduce the coding gene of ZmST1 protein into a receptor plant.
- 9. The method according to any one of claims 6-8, wherein: the coding gene sequence of the ZmST1 protein is shown as a sequence 2 in a sequence table.
- 10. The method according to any one of claims 6-9, characterized in that: the plant is a dicotyledon or monocotyledon; and/or, the monocot is maize.
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