CN113980106B - Small peptide for regulating and controlling sizes of plant seeds and organs, and coding gene and application thereof - Google Patents

Abstract

The invention discloses a small peptide for regulating the size of plant seeds and organs, its coding gene and application. The invention specifically discloses the application of the polypeptide AtZSP1 (AtZSP1 small peptide) whose amino acid sequence is SEQ ID No. 1 and its encoding gene in regulating the size of plant seeds and organs. The present invention uses the T-DNA insertion mutant library, adopts the method of reverse genetics, and according to bioinformatics analysis, successfully identifies a gene involved in the regulation of Arabidopsis seed and organ size for the first time, named AtZSP1 gene, and through the gene Engineering methods increase the size of plant seeds and/or organs, and obtain AtZSP1 overexpression plants, enriching high-quality genetic resources for the development and application of small peptides, which can be well applied to the improvement of crop varieties and increase crop yield , which has important practical value for plant breeding.

Description

Small peptides regulating plant seed and organ size and their encoding genes and applications

technical field

The invention belongs to the field of biotechnology, and in particular relates to small peptides for regulating the size of plant seeds and organs, coding genes and applications thereof.

Background technique

Seed size is one of the main factors affecting crop yield, directly determines crop yield, and has long been an important goal of many crop breeding and improvement. In addition, the size of plant organs is also an important trait in sciences such as agronomy, horticulture, and herbalism. Therefore, the study of the regulatory mechanism of seed and organ size has become an important research field in plant science. The size of plant organs is determined by both cell proliferation and cell expansion, the former increases cell number through cell division, and the latter increases cell size through cell growth. Many genes involved in the regulation of cell proliferation and cell growth, thereby affecting plant organ size have been identified (Zhang X, Guo W, Du D, Pu L, Zhang C. Ove reexpression of a maize BR transcription factor ZmBZR1 in Arabidopsis enlarge ges organ and seed size of the transgenic plants. Plant Sci. 2020; 292:110378.). Among these genes, a class of small peptides is also involved in this process. For example, ROTUNDIFOLIA4 (ROT4) regulates cell proliferation at the top of Arabidopsis stems. Overexpression of ROT4 reduces the size of meristems, thereby reducing leaves (Ikeuch i M, Yamaguchi T, Kazama T, Ito T, Horiguchi G, Tsukaya H. ROTUNDIFOLIA4 regulates cell proliferation along the body axis in Arabidopsis shoot. Plan t Cell Physiol. 2011; 52(1):59-69.). Phytosulfokine-α (PSK-α) affects root growth by regulating cell expansion, and PSK receptor gene Atpskr1-T mutants shorten root length mainly due to reduced cell size (Kutschmar A, Rzewuski G, Stuhrwohldt N, Beemster GT , Inze D, Saute r M. PSK-alpha promotes root growth in Arabidopsis. New Phytol. 2009; 181(4):820-31.). Although many genes affecting plant organ size have been identified, studies on their genetics and molecular biological mechanisms are still limited.

Small peptides broadly refer to proteins with a length of less than 250 amino acids, but it is also defined as 5-60 amino acids in length (Matsubayashi Y. Posttranslationally modified small-peptide signals in plants. Annu Rev Plant Biol .2014;65:385-413.). Small peptides are involved in many processes such as cell proliferation, root system development, pollen fertility, stomatal switch, absorption and regulation of mineral elements, growth and development against pests and diseases, and responses to abiotic stress. According to the characteristics of whether there is a signal peptide sequence at the N-terminus, small peptides can be mainly divided into two categories: secreted, that is, small peptides that are secreted to the outside of the cell (the N-terminal contains a signal peptide); non-secreted, that is, in the cell Functional small peptide (no signal peptide at the N-terminus). At present, many small peptides have been identified to participate in the growth and development of plants, such as CLAVATA3 regulates the size of SAM (Rojo E, Sharma V K, Kovaleva V, et al. CLV3 Is Localized to the Extracellular Space, Where It Activates the Arabidopsis CLAVATA Stem Cell Signaling Pathway [J].Plant Cell,2002,14(5):969-977.); LURE guides the pollen tube to the embryo sac (Okuda S, Tsutsui H, Shiina K, Sprunck S, Takeuchi H, Yui R, et al. Defensin-likepolypeptide LUREs are pollen tube attractants secreted from synergidcells.Nature.2009; 458(7236):357-61.); RALFs protein family plays an important role in plant vegetative growth and reproductive growth, etc. (Bedinger PA, Pearce G, Covey PA. RALFs: peptide regulators of plant growth. Plant Signal Behav. 2010; 5(11): 1342-6.; Qu LJ, Li L, Lan Z, Dresselhaus T. Peptide signaling during the pollen tube journey and double fertilization. J Exp Bot. 2015;66(17):5139-50.). However, there are still a large number of small peptides with unknown functions that have not been identified in plants, and the development and application of small peptides are greatly limited due to the large number of small peptides in plants and the difficulty in studying their biological functions. Studies have shown that based on the comprehensive method of "bioinformatics prediction-mutant identification-gene function identification", not only small peptides can be effectively identified but also the biological functions of small peptides can be verified. The low abundance brings obstacles to the identification of small peptides, and at the same time avoids the disadvantages of cumbersome, time-consuming, and high technical requirements of traditional biochemical analysis techniques.

The development and application of plant small peptides is an emerging and promising research field. There are still a large number of small peptides in plants to be discovered. Just like human beings' understanding of soil microorganisms, there are more gaps waiting to be filled. Therefore, it is of great significance to identify unknown small peptides in plants and explore their functions.

Contents of the invention

The technical problem to be solved by the present invention is how to regulate the size of plant seeds and/or organs.

In order to solve the above-mentioned technical problems, the present invention firstly provides the application of polypeptides that regulate the size of plant seeds and/or organs or substances that regulate the activity or content of the polypeptides, and the applications can be any of the following:

D1) Application of a polypeptide or a substance that regulates the activity or content of the polypeptide in regulating the size of plant seeds and/or organs;

D2) Application of a polypeptide or a substance that regulates the activity or content of the polypeptide in the preparation of a product that regulates the size of plant seeds and/or organs;

D3) Use of polypeptides or substances regulating the activity or content of said polypeptides in the cultivation of plants with increased or decreased seed and/or organ size;

D4) Use of polypeptides or substances regulating the activity or content of said polypeptides in the production of products for cultivating plants with increased or decreased seed and/or organ size;

D5) Application of polypeptides or substances regulating the activity or content of said polypeptides in plant breeding;

The name of the polypeptide is AtZSP1, which can be any of the following:

A1) The amino acid sequence is the polypeptide of SEQ ID No.1;

A2) Substituting and/or deleting and/or adding more than one amino acid residue to the amino acid sequence shown in SEQ ID No.1 has more than 80% identity with the polypeptide shown in A1) and has the ability to regulate plant seeds and/or organ size functional polypeptides;

A3) A fusion polypeptide obtained by linking a tag at the N-terminal and/or C-terminal of A1) or A2).

A4) The amino acid sequence is a polypeptide of SEQ ID No.8;

A5) Substituting and/or deleting and/or adding more than one amino acid residue to the amino acid sequence shown in SEQ ID No.8 has more than 80% identity with the polypeptide shown in A4) and has the ability to regulate plant seeds and/or organ size functional polypeptides;

A6) A fusion polypeptide obtained by linking a tag at the N-terminal and/or C-terminal of A4) or A5).

In order to facilitate the purification of the polypeptide, the amino terminus and/or carboxyl terminus of the polypeptide can be linked with tags as shown in Table 1.

Table 1: Sequence of tags

Label Residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

The AtZSP1 polypeptide in the above A2) may be a polypeptide having 75% or more identity with the amino acid sequence of the polypeptide shown in SEQ ID No. 1 and having the same function. Said having 75% or more identity means having 75%, having 80%, having 85%, having 90%, having 95%, having 96%, having 97%, having 98% or having 99% identity .

The AtZSP1 polypeptide in the above A2) can be synthesized artificially, or its coding gene can be synthesized first, and then biologically expressed.

The AtZSP1 polypeptide in A2) above may be a small peptide consisting of 57 amino acids. The small peptide is named AtZSP1 small peptide.

The coding gene of the AtZSP1 polypeptide in the above A2) can be obtained by deleting the codon of one or several amino acid residues in the DNA sequence shown in SEQ ID No.2, and/or carrying out one or several base pairs of missense mutation, and/or link the coding sequence of the tag shown in Table 1 at its 5' end and/or 3' end.

Wherein, the DNA molecule shown in SEQ ID No.2 encodes the AtZSP1 polypeptide shown in SEQ ID No.1.

The present invention also provides the application of biological materials related to the AtZSP1 polypeptide, and the application can be any of the following:

E1) Application of biological materials related to AtZSP1 polypeptide in regulating plant seed and/or organ size;

E2) Application of biological materials related to AtZSP1 polypeptide in the preparation of products for regulating the size of plant seeds and/or organs;

E3) Use of biological materials related to AtZSP1 polypeptides for cultivating plants with increased or decreased seed and/or organ size;

E4) Use of biological materials related to AtZSP1 polypeptides for the production of products for cultivating plants with increased or decreased seed and/or organ size;

E5) Application of biological materials related to AtZSP1 polypeptide in plant breeding;

The biological material can be any of the following:

B1) a nucleic acid molecule encoding an AtZSP1 polypeptide;

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);

D1) Nucleic acid molecules that inhibit or reduce the expression of genes encoding AtZSP1 polypeptides;

D2) an expression cassette containing the nucleic acid molecule of D1);

D3) a recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2);

D4) A recombinant microorganism containing the nucleic acid molecule described in D1), or a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism containing the recombinant vector described in D3);

D5) a transgenic plant cell line containing the nucleic acid molecule described in D1), or a transgenic plant cell line containing the expression cassette described in D2);

D6) a transgenic plant tissue containing the nucleic acid molecule described in D1), or a transgenic plant tissue containing the expression cassette described in D2);

D7) A transgenic plant organ containing the nucleic acid molecule described in D1), or a transgenic plant organ containing the expression cassette described in D2).

In the above application, the nucleic acid molecule described in B1) can be a DNA molecule as shown in b1) or b2) or b3) or b4) as follows:

b1) the coding sequence (CDS) is a DNA molecule or a cDNA molecule shown in SEQ ID No.2;

b2) the nucleotide sequence is a DNA molecule or a cDNA molecule shown in SEQ ID No.2;

b3) A DNA molecule or cDNA molecule that has 75% or more identity to the nucleotide sequence defined in b1) or b2) and encodes the AtZSP1 polypeptide;

b4) A DNA molecule or a cDNA molecule that hybridizes to the nucleotide sequence defined in b1) or b2) under stringent conditions and encodes the AtZSP1 polypeptide.

Those skilled in the art can easily use known methods, such as directed evolution or point mutation methods, to mutate the nucleotide sequence encoding the AtZSP1 polypeptide of the present invention. Those artificially modified nucleotides with 75% or higher identity to the nucleotide sequence of the isolated AtZSP1 polypeptide of the present invention, as long as they encode the AtZSP1 polypeptide and have the function of the AtZSP1 polypeptide, are all derived from the core of the present invention. Nucleotide sequence and is equivalent to the sequence of the present invention.

In the above application, the stringent conditions can be as follows: 50°C, hybridization in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4 and 1mM EDTA, at 50°C, 2×SSC, 0.1% SDS Rinse in medium; can also be: 50°C, hybridize in a mixed solution of 7% SDS, 0.5M NaPO4 and 1mM EDTA, rinse in 50°C, 1×SSC, 0.1% SDS; can also be: 50°C, in 7 Hybridization in a mixed solution of %SDS, 0.5M NaPO4 and 1mM EDTA, rinse at 50°C, 0.5×SSC, 0.1% SDS; also: 50°C, in a mixed solution of 7% SDS, 0.5M NaPO4 and 1mM EDTA Hybridization, wash at 50°C, 0.1×SSC, 0.1% SDS; alternative: 50°C, hybridization in a mixed solution of 7% SDS, 0.5M NaPO4 and 1mM EDTA, at 65°C, 0.1×SSC, 0.1% Rinse in SDS; can also be: in 6×SSC, 0.5% SDS solution, hybridize at 65°C, then wash the membrane once with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS; also can For: in 2×SSC, 0.1% SDS solution, hybridize at 68°C and wash the membrane twice, 5 min each time, and in 0.5×SSC, 0.1% SDS solution, hybridize at 68°C and wash the membrane for 2 times Each time, 15 minutes each time; it can also be: 0.1×SSPE (or 0.1×SSC), 0.1% SDS solution, hybridization at 65°C and washing the membrane.

The identity of 75% or more may be 80%, 85%, 90% or more.

In the above applications, identity refers to the identity of amino acid sequence or nucleotide sequence. 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 Lambdaratio to 11 respectively , 1 and 0.85 (the default value) and search for the identity of a pair of amino acid sequences for calculation, and then the value (%) of the identity can be obtained.

In the above application, the above 80% identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

In the above application, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

In the above application, the substance that regulates the activity or content of the polypeptide may be a substance that regulates the expression of a gene encoding the AtZSP1 polypeptide.

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 polypeptide translated by the gene regulation).

The substance for regulating gene expression can specifically be the biological material described in any one of B1)-B3) and D1)-D3).

In the above-mentioned application, the expression cassette (AtZSP1 gene expression cassette) described in B2) refers to the DNA capable of expressing the AtZSP1 polypeptide in the host cell. the terminator. 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. Examples of promoters include, but are not limited to: Cauliflower Mosaic Virus Constitutive Promoter 35S: Wound-Inducible Promoter from Tomato, Leucine Aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxy acid S-methyl ester)); tomato Protease inhibitor II promoter (PIN2) or LAP promoter (both inducible with methyl jasmonate); heat shock promoter (US Patent 5,187,267); tetracycline-inducible promoter (US Patent 5,057,422) ; Seed-specific promoters, such as millet seed-specific promoter pF128 (CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (for example, the promoters of phaseolin, napin, oleosin and soybean beta conglycin (Beachy et al. (1985) EMBO J. 4:3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are cited in their entirety. Suitable transcription terminators include, but are not limited to: Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcSE9 terminator, and nopaline and octopine synthases. Enzyme terminators (see, for example: Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) ) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) PlantCell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627).

An existing expression vector can be used to construct a recombinant vector containing the AtZSP1 gene expression cassette. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Company), etc. The plant expression vector may also include the 3' untranslated region of the foreign gene, that is, the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide polyadenylic acid to be added to the 3' end of the mRNA precursor, such as Agrobacterium crown gall tumor induction (Ti) plasmid gene (such as nopaline synthase gene Nos), plant gene (such as soybean The untranslated region transcribed at the 3′ end of the storage protein gene) has similar functions. When using the gene of the present invention to construct plant expression vectors, enhancers can also be used, including translation enhancers or transcription enhancers, and these enhancer regions can be ATG initiation codons or adjacent region initiation codons, etc. The reading frames of the sequences are identical to ensure correct translation of the entire sequence. The sources of the translation control signals and initiation codons are extensive and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes (GUS gene, luciferase gene, etc.) genes, etc.), antibiotic marker genes (such as the nptII gene that confers resistance to kanamycin and related antibiotics, the bar gene that confers resistance to the herbicide phosphinothricin, and the hph gene that confers resistance to the antibiotic hygromycin , and the dhfr gene that confers resistance to methotrexate, the EPSPS gene that confers resistance to glyphosate) or the chemical resistance marker gene (such as the herbicide resistance gene), the mannose-6- that provides the ability to metabolize mannose Phosphate isomerase gene. Considering the safety of the transgenic plants, the transformed plants can be screened directly by adversity without adding any selectable marker gene.

In the above application, the vector can be a plasmid vector, a cosmid vector, a phage vector, a virus vector or an artificial minichromosome vector.

In the above applications, the microorganisms can be yeast, bacteria, algae or fungi. Among them, the bacteria can be from Escherichia (Escherichia), Erwinia (Erwinia), Agrobacterium (Agrobacterium), Flavobacterium (Flavobacterium), Alcaligenes (Alcaligenes), Pseudomonas (Pseudomonas), Bacillus (Bacillus) and so on. Specifically, the bacteria can be Escherichia coli.

The polypeptides of the present invention include peptides, polypeptides or small peptides, whether naturally occurring or synthetic.

The present invention also provides a method for increasing the size of plant seeds and/or organs, the method comprising increasing the size of plant seeds and/or organs by increasing or increasing the expression and/or activity of the AtZSP1 polypeptide in the plant.

In the above method, said improving or increasing the expression and/or activity of the AtZSP1 polypeptide in the plant is achieved by improving or increasing the expression level of the gene encoding the polypeptide in the plant.

Further, said improving or increasing the expression and/or activity of the AtZSP1 polypeptide in the plant may be overexpression of the gene encoding the AtZSP1 polypeptide in the plant.

Further, the method includes the following steps:

M1) Constructing an overexpression vector containing the gene encoding the AtZSP1 polypeptide;

M2) The overexpression vector described in M1) is directly introduced into the recipient plant or mediated by Agrobacterium into the recipient plant to obtain a transgenic plant containing the gene encoding the AtZSP1 polypeptide overexpressed.

The overexpression vector contains a strong promoter that drives the overexpression of the coding sequence for the AtZSP1 polypeptide in recipient plants.

The strong promoter can be a 35s promoter, or more than two 35s promoters, or a 35s promoter plus a Ubi promoter.

Further, in one embodiment of the present invention, the plant is Arabidopsis.

The present invention also provides a method for reducing the size of plant seeds and/or organs, the method comprising reducing the size of plant seeds and/or organs by inhibiting or reducing the expression of the gene encoding the AtZSP1 polypeptide and/or the activity of the AtZSP1 polypeptide in the plant.

In the above method, the method for inhibiting or reducing the expression of the gene encoding the AtZSP1 polypeptide and/or the activity of the AtZSP1 polypeptide in the plant may be to delete nucleotides 31-58 of SEQ ID No. 2 in the plant genome.

The present invention also provides the use of a method for increasing or decreasing the size of seeds and/or organs of a plant for the cultivation of plants having increased or decreased size of seeds and/or organs.

The present invention also provides the use of methods for increasing or decreasing the size of seeds and/or organs of plants in plant breeding.

The plant breeding may be transgenic breeding to increase or decrease crop seed and/or organ size.

Said modulating plant seed and/or organ size may be increasing or decreasing plant seed and/or organ size.

In the above, the plant may be any of the following:

F1) monocot or dicot;

F2) Plants of the order Lilyaceae, plants of the order Graminees or plants of the order Beans;

F3) Brassicaceae, grasses or leguminous plants;

F4) Arabidopsis, Brassica, Zea, Oryza, Setaria, Sorghum or Glycine;

F5) Arabidopsis, rapeseed, Chinese cabbage, corn, rice, millet, sorghum or soybean.

The present invention utilizes the T-DNA insertion mutant library, adopts the method of reverse genetics, and according to bioinformatics analysis, classifies unknown small peptide proteins of Arabidopsis thaliana, and performs a single-copy gene analysis on its amino acid length less than 100. During identification, a gene encoding 57 amino acids was found, and the small peptide gene was named AtZSP1. The T-DNA insertion mutant of the gene was identified, and the homozygous mutant was screened out. And using CRISPR/Cas9 technology to obtain another homozygous mutant, both mutants are complete knockout mutants. Using the idea of reverse genetics to conduct a series of gene function research on homozygous mutants. The mutant phenotype produced by gene knockout is the phenotype after the gene is deleted, thus directly indicating the relationship between the function of the gene and the phenotype, so that the function of the gene can be directly and quickly detected.

The present invention observes the phenotype of the homozygous mutant, and the results show that the seed size and 100-grain weight of the mutant are significantly smaller than that of the wild type, and the plant is short, and the size of the leaves, the number of flower buds and the number of siliques on the main stem are all lower than those of the wild type. less than wild type. It shows that the deletion of AtZSP1 gene affects the seed and organ size of Arabidopsis. In addition, the phenotype of the obtained homozygous overexpression plants was observed, and it was found that the phenotype of the overexpression plants was completely opposite to that of the mutant. Since the size of plant organs is determined by the number of cells and the size of the cells, the number and size of the cotyledon cells of the wild type and mutants were counted. The average cell size of the cotyledons was also smaller than that of the wild type. It shows that AtZSP1 gene may affect the size of organs by controlling cell proliferation and cell expansion. In addition, the polypeptide encoded by this gene has homologous proteins in crops such as rapeseed, corn, rice, Chinese cabbage, soybean, millet, and sorghum, and the sequences of these proteins are relatively conserved in these species, suggesting that AtZSP1 plays a role in crop variety improvement. There are good prospects.

The present invention successfully identified a small AtZSP1 peptide for the first time, cloned the AtZSP1 gene, verified the gene function, and increased the size of plant seeds and/or organs through genetic engineering methods, and obtained AtZSP1 overexpressed plants, which is the development of small peptides It has enriched high-quality genetic resources in the field of application and application, which can be well applied to the improvement of crop varieties, increase crop yield, and has important practical value for plant breeding.

Description of drawings

Figure 1 is the electrophoresis diagram for identification of atzsp1-1 homozygous mutant.

Fig. 2 is a schematic diagram of the structure of the AtZSP1 gene and the T-DNA insertion site. The atzsp1-1 mutant is T-DNA inserted in the first exon of AtZSP1 gene at 27bp away from ATG.

Figure 3 is a schematic diagram of the selection and mutation form of the atzsp1-2 mutant target constructed by CRISPR/Cas9 technology. Mutant atzsp1-2 is a homozygous mutation with a 28bp deletion between the two target sites, resulting in the premature termination of the stop codon TGA after 118bp, leading to early termination of protein translation.

Figure 4 is the atzsp1-2 mutant without cas9 vector. 1/2MS Medium means growth on 1/2MS medium, 1/2MS+Hyg Medium means growth on hygromycin-containing medium.

Fig. 5 is a graph showing the analysis results of AtZSP1 gene expression in the mutant. Col indicates wild-type Arabidopsis control.

Fig. 6 is a graph showing the results of leaf and fresh weight analysis of mutants and complementary plants.

Figure 7 is a schematic diagram of the construction of a complementary cloning vector. Among them, pCAMBIA1300 is the basic vector; gAtZSP1 (1948bp) is the full-length AtZSP1 gene, including a 1184bp promoter, a 25bp 5' non-coding sequence, a 278bp gene coding sequence (exons + introns) and a 461bp 3'non-coding sequence. Coding sequence; BamHI and PstI are two single enzyme cutting sites used in the construction of complementary vectors; Kan means kanamycin, which is the selection gene for antibiotic kanamycin resistance.

Figure 8 is a schematic diagram of the construction of the overexpression vector.

Fig. 9 is a diagram showing the results of analysis of AtZSP1 gene expression in mutants and overexpression plants.

Fig. 10 is a graph showing the results of analysis of seed size and 100-grain weight of mutants and overexpression plants.

Figure 11 is a graph showing the results of mature embryo analysis of mutants and overexpression plants.

Figure 12 is a graph showing the leaf analysis results of mutants and overexpression plants grown in soil for 17 days.

Fig. 13 is a graph showing the analysis results of plant height, flower bud number and silique number of mutants and overexpression plants.

Figure 14 is a graph showing the results of cotyledon cell number and cell size analysis of mutants.

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 wild-type Arabidopsis thaliana (WT, Col) in the following examples is the Colombian ecotype Arabidopsis thaliana (Col-0), Arabidopsis Biological Resource Center (ABRC).

The pCBC-DT1T2 in the following examples was donated by the laboratory of Professor Chen Qijun of China Agricultural University (Lei Zhu, Liang-Cui Chu, Yan Liang, Xue-Qin Zhang, Li-Qun Chen, De Ye1. The Arabidopsis CrRLK1L protein kinases BUPS1 and BUPS2 are required for normal growth of pollen tubes in the pistil. The Plant Journal (2018) 95, 474–486), the public can obtain from China Agricultural University, to repeat the experiment of the present invention.

The pHEC401 in the following examples was donated by the laboratory of Professor Chen Qijun of China Agricultural University (Lei Zhu, Liang-Cui Chu, Yan Liang, Xue-Qin Zhang, Li-Qun Chen, De Ye1. The Arabidops is CrRLK1L protein kinases BUPS1 and BUPS2 are required for normal growth of pollen tubes in the pistil. The Plant Journal (2018) 95, 474–486), the public can obtain from China Agricultural University, to repeat the experiment of the present invention.

The pSuper1300 in the following examples was donated by Gong Zhizhong Laboratory of China Agricultural University (Yanglin Ding, Jian Lv, Yiting Shi, Junping Gao, Jian Hua, Chunpeng Song, Zhizhong Gong, ShuhuaYang. EGR2 phosphatase regulates OST1 kinase activity and freezing tolerance in Arabidopsis. The EMBO Journal (2019) 38:e99819), the public can obtain from China Agricultural University, to repeat the experiments of the present invention.

Example 1 Screening of atzsp1 mutants and identification of AtZSP1 gene

1. Screening of atzsp1 mutants

The invention firstly screens and classifies unknown small peptides with amino acid length less than 150 in Arabidopsis thaliana, and there are 3698 unidentified genes in total. In order to obtain the efficiency of effective mutants, the scope was further narrowed, and a single-copy gene with an amino acid length of less than 100 was identified, and the gene inserted in the exon was ordered from the official website of TAIR (http://www.arabidopsi s.org/). T-DNA insertion mutants. After the homozygous mutants were screened by PCR, a preliminary phenotype observation was carried out, and a gene involved in the regulation of Arabidopsis seed and organ size was identified, named AtZSP1 gene. The amino acid sequence encoded by the AtZSP1 gene is the polypeptide AtZSP1 shown in SEQ ID No. 1 in the sequence listing. The genomic gene of AtZSP1 is the double-stranded DNA shown in SEQ ID No.3 in the sequence table, the 1st-1184th nucleotide is the promoter, the 1185-1209th nucleotide is the 5'UTR, and the 1210th- Nucleotide 1277 is the first exon, nucleotides 1278-1381 is the first intron, nucleotides 1382-1487 is the second exon, nucleotides 1488-1948 for the 3'UTR. The cDNA gene of AtZSP1 is a double-stranded DNA shown in SEQ ID No. 4 in the sequence listing, and the 26th-199th nucleotides are the coding sequence (CDS).

After AtZSP1 gene mutation, the seed, leaf, root length, inflorescence and plant height were all smaller than the wild type, while the seeds, leaf length, root length, inflorescence and plant height of the overexpressed plants were larger than the wild type. In addition, through the search of plant genome sequences and bioinformatics research, there is no protein homologous to AtZSP1 in Arabidopsis, and its protein sequence is highly conserved in crops such as corn, rice, rape, Chinese cabbage and soybean, which means that The AtZSP1 genes identified in the present invention are broadly applicable to other plant species in which orthologous AtZSP1 genes with similar functions exist.

2. Identification of atzsp1 mutant and AtZSP1 gene

(1) atzsp1-1 mutant

The T-DNA insertion mutant (SALK_11106C) ordered from the TAIR official website was named atzsp1-1, and the mutant was identified by PCR with two specific primers on the AtZSP1 gene and specific primers on the T-DNA, and the results showed that , the mutant is a homozygous mutant (Figure 1). In Fig. 1, LP and RP are primers on AtZSP1 gene, and LBb1.3 is a specific primer on T-DNA. Sequencing results showed that T-DNA was inserted at 27 bp away from ATG on the first exon of AtZSP1 gene (Fig. 2).

(2) atzsp1-2 mutant

Using CRISPR/Cas9 technology to construct the second mutant, the specific process is: first construct a dual-target vector, and select two sequences at the 24-46bp position and 53-75bp position from the start codon ATG on the gene Targets, that is, the sequences shown in Target1 and Target2 in FIG. 3 . The sequence of Target1 is: CCAAACAGTGGCAATCTCCGGCG, and the sequence of Target2 is CCGCCGTCTCATGCTGGTGAGTG. Using pCBC-DT1T2 as a template, use primers to amplify the DNA encoding Target1-Target2 by PCR, recover the obtained PCR product (the sequence of the PCR product is shown in SEQ ID No.5), and add The PCR product, pHEC401 vector, BsaI restriction endonuclease and T4 Ligase ligase were digested and ligated, and the ligated product was transferred into Escherichia coli competent DH5α cells. After colony PCR and sequencing, the correctly ligated plasmid was named as pHEC401-ZSP1-cas9. pHEC401-ZSP1-cas9 replaces the fragment between the two BsaI recognition sites of pHEC401 with the DNA fragment shown in the 13th to 614th positions of SEQ ID No.5, keeping the other sequences of pHEC401 unchanged, and obtains expression targeting Recombinant expression vectors of sgRNA1 and sgRNA2 and cas9 of Target1 and Target2.

The pHEC401-ZSP1-cas9 was transformed into Agrobacterium tumefaciens GV3101, and the correctly identified recombinant Agrobacterium was named GV-ZSP1-cas9. GV-ZSP1-cas9 was transformed into wild-type Arabidopsis inflorescence by Agrobacterium dipping method; the seeds (T0 generation seeds) of the harvested T0 generation transgenic plants were screened on the medium containing hygromycin, and the selected The positive seedlings of the T1 generation were sequenced to analyze the mutant form of the AtZSP1 gene. A homozygous mutation with a deletion of 28bp between the two targets was identified, which resulted in the appearance of the stop codon TGA ahead of time after 118bp (the sequence before the deletion), which caused the early termination of protein translation. The mutant was named atzsp1-cas9 ( image 3). In order to eliminate the CRISPR/Cas9 vector in the mutant, the atzsp1-cas9 mutant was selfed for one generation, and the vector-free mutant was isolated from the progeny plants. 50 μg/mL hygromycin) could not grow, indicating that the carrier had been cleared, and the mutant was named atzsp1-2 (Figure 4). The nucleotide sequence of the AtZSP1 mutant gene in atzsp1-cas9 is shown in SEQ ID No.6, the coding sequence is shown in 1-93 nucleotides of SEQ ID No.7, and the amino acid sequence of the AtZSP1 mutant polypeptide is shown in SEQ ID No. .8 shown.

(3) Expression analysis

The expression levels of two homozygous mutants, atzsp1-1 and atzsp1-2, were analyzed: RNA from wild-type Arabidopsis and seedlings of the two mutants grown under light for 10 days were extracted, reverse-transcribed into cDNA, and used Primer (5'-CGTTCTCGTTGATCCAAACAG-3') and primer (5'-GTGGAATCAGAATTGGAGCCT-3') were used for qRT-PCR analysis. Taking the expression level of wild-type Arabidopsis as 1, the results showed that the expression of the AtZSP1 gene was not detected in the two mutants, indicating that the two mutants were complete knockout mutants ( FIG. 5 ).

3. Cloning of AtZSP1 gene and complementation experiment of atzsp1-1 mutant

Preliminary observations of the two mutants, atzsp1-1 and atzsp1-2, have the same phenotype, and both have small leaf phenotypes. The fresh weight of the aboveground part and the surface size of the leaves were measured for the seedlings grown in the soil for 17 days. The results showed that the mutants The fresh weight of leaves and the size of the leaf surface were significantly smaller than those of the wild type (Fig. 6). In order to confirm that the phenotype of the atzsp1-1 mutant is caused by the deletion of the AtZSP1 gene, an anaplerotic clone of the atzsp1-1 mutant was constructed ( FIG. 7 ), and an anaplerotic experiment was performed.

The specific process is:

(1) Using the genomic DNA of wild-type Arabidopsis (the full length of the AtZSP1 gene includes a 1184bp promoter, a 25bp 5' non-coding sequence, a 278bp gene sequence and a 461bp 3' non-coding sequence) as a template, use the AtZSP1 gene Specific primers (containing BamHI and PstI two restriction endonuclease sites respectively) (5'-CGG GGATCCAAACGGGCATCGGTATGAAG-3' and 5'-CGA CTGCAG TTTCCCACATAGGTACAT ACA-3') were used for PCR amplification, and the PCR products were purified Recovered and ligated to the pCAMBIA1300 vector after enzyme digestion, and the recombinant anaplerotic expression vector that was cloned and sequenced correctly was named pAtZSP1::AtZSP1 ( FIG. 7 ). pAtZSP1::AtZSP1 is the recombinant expression obtained by replacing the fragment (small fragment) between the BamHI and PstI recognition sites of pCAMBIA1300 with the DNA fragment shown in SEQ ID No.3 in the sequence list, keeping other sequences of pCAMBIA1300 unchanged carrier.

The recombinant plasmid pAtZSP1::AtZSP1 was transformed into Agrobacterium GV3101 by electric shock method, and the correctly identified recombinant Agrobacterium was named GV-pAtZSP1::AtZSP1. Transgenic Arabidopsis thaliana seeds of the T0 generation were obtained by transforming the atzsp1-1 mutant into the atzsp1-1 mutant through the method of inflorescence mediated by Agrobacterium.

(2) Sow the T0 transgenic Arabidopsis seeds obtained in step (1) on a medium containing hygromycin (1/2MS+50μg/mL hygromycin), and the Arabidopsis that can grow normally is T1 Transgenic positive plants of the T1 generation, and the seeds harvested from the positive transgenic plants of the T1 generation are T1 generation transgenic Arabidopsis seeds.

For a certain T1 generation plant, if the following two conditions are met, the T1 generation plant is a single-copy inserted transgenic plant: ① The T1 generation plant is a hygromycin-resistant plant; ② The T1 generation plant is obtained by selfing Among the T2 generation plants, the number ratio of hygromycin-resistant plants to hygromycin-sensitive plants was basically 3:1.

For a certain T2 generation plant, if the following three conditions are met, the T2 generation plant and its selfed offspring are a homozygous transgenic line: ① The T2 generation plant is a hygromycin-resistant plant; ② Its T1 The generation plants were transgenic plants with single copy insertion; ③The T3 generation plants sampled and tested were all hygromycin-resistant plants.

66 transgenic positive plants of the T1 generation were identified by PCR, and single-copy plants were screened in the T2 generation, and the seeds of these single-copy plants were harvested. ) for homozygous screening, and a total of 9 homozygous anaplerotic plants were screened, two of which were named as atzsp1-comp-1 and atzsp1-comp-2 in T2 generation. SPSS11.5 statistical software was used to process the data, and the experimental results were expressed as mean ± standard deviation, using t-test test, P<0.01 (**) indicated that there was a significant difference compared with wild-type Arabidopsis, P< 0.001 (***) indicates extremely significant difference compared with wild-type Arabidopsis. Through phenotype observation, it was found that the leaf size of the replenished plants was more consistent with that of the wild type. The fresh weight of the aboveground part and the surface size of the largest leaf were measured for the seedlings cultured in the soil for 17 days, and the size also returned to the wild type level. It shows that the introduction of AtZSP1 gene into the atzsp1-1 mutant plants can complement the small organ phenotype of the atzsp1-1 mutant, and the small organ phenotype of the atzsp1-1 mutant is indeed caused by the deletion of the AtZSP1 gene ( FIG. 6 ).

The acquisition of embodiment 2 AtZSP1 gene overexpression plant

In order to further study the function of AtZSP1 gene, the overexpression plants of AtZSP1 gene were constructed.

The specific process is:

(1) Construction of overexpression vector

Using the cDNA of wild-type Arabidopsis as a template, PCR was performed with primers 5'-CGA CTGCAG ATGCAAAAATCGTTCTCGTTG-3' and 5'-GAG ACTAGT GGAATGGGGAGTGGAATCAG-3' (respectively containing two restriction enzyme sites, PstI and SpeI) For amplification, the PCR product was purified and recovered, digested with PstI and SpeI, and then ligated to the pSuper1300 vector (Figure 8). The ligation product was transformed into Escherichia coli competent DH5α cells, cultured on LB solid medium containing 50 μg/mL kanamycin, and sequenced after colony PCR to obtain the correct clone. The recombinant overexpression vector was named p35S::AtZSP1. p35S::AtZSP1 replaces the fragment (small fragment) between the PstI and SpeI recognition sites of the pSuper1300 vector with the DNA fragment shown in SEQ ID No.2 in the sequence table (remove TGA), keeping other sequences of the pSuper1300 vector unchanged Change, the obtained AtZSP1 gene recombinant expression vector. The p35S::AtZSP1 was transformed into Agrobacterium GV3101, and the correctly identified recombinant Agrobacterium was named GV-p35S::AtZSP1.

(2) Acquisition of AtZSP1 gene overexpression plants

Take 3 mL of cultured GV-p35S::AtZSP1 (recombinant Agrobacterium containing overexpression vector) Agrobacterium liquid and transfer it into 150 mL of three-antibody LB medium (Rif+Gen+Kan), overnight at 28°C and 250rpm Cultivate until orange, so that the OD600nm value of the bacterial solution is 1.6-1.8; transfer the bacterial solution to a centrifuge bottle, and centrifuge at 3,000 rpm for 15 minutes at 4°C; discard the supernatant, and add 1/2 MS culture solution (0.22g MS powder and 2g sucrose , add water to dissolve, adjust the pH to 5.8, dilute to 100mL with ddH ₂ O, add 12ml of Silwet L-77 reagent, stir evenly), suspend the bacterial precipitate, and adjust the pH to 0.6-0.8; cut before dipping For siliques, place the inflorescences of wild-type Arabidopsis plants in full flowering stage in a petri dish filled with bacterial solution and soak for 10s; place the soaked plants flat on a tray, and cultivate them in a dark environment for 18-24 hours Return to normal light cultivation. The seeds produced by T0 generation transgenic plants (p35S::AtZSP1 transgenic plants) (T0 generation seeds) were collected. The plants grown from the seeds of the T0 generation are the T1 generation, and so on, and T2 and T3 represent the second and third generation of transgenic plants, respectively.

After sterilizing the T0 generation seeds, spread them on 1/2MS+50 μg/mL hygromycin solid medium (Murashige and Skoog basal medium, 0.8% agar powder, pH5.8, 50 μg/mL hygromycin), and treat at 4 °C On 2d, move to 22°C, cultivate under light (16h light/8h dark) conditions for 8-10d, pick resistant plants and plant them in soil, and continue to cultivate under light conditions. Genomic DNA of leaves was extracted, primers (F: 5'-ATGCAAAATCGTTCTCGTTG-3' and R: 5'-CTTATCGTCATCGTCCTTGT-3') were used to amplify the AtZSP1-Flag fragment by PCR, and the positive plants were further determined through PCR identification (PCR product approx. 240bp).

(3) Identification of AtZSP1 gene overexpression plants

The total RNA of wild-type Arabidopsis, atzsp1-1 and p35S::AtZSP1 transgenic seedlings was extracted by Trizol method, cDNA was synthesized by reverse transcription (refer to the instruction of Genestar reverse transcription kit), and stored at -20°C for future use. The expression level was detected by real-time fluorescent quantitative PCR (qRT-PCR). Amplification was carried out with primer 1 and primer 2 specific to the coding region of AtZSP1 gene.

Primer 1: 5'-CGTTCTCGTTGATCCAAACAG-3'

Primer 2: 5'-GTGGAATCAGAATTGGAGCCT-3'

The PCR reaction system is as follows:

2X power SYBR Green PCR Master Mix: 10μL;

Primer 1 (10 μmol/L): 0.2 μL;

Primer 2 (10 μmol/L): 0.2 μL;

cDNA: 1 μL;

ddH2O: 8.6 μL.

Real-time PCR reaction program: pre-denaturation at 95°C for 5min; 40 cycles of 94°C for 25sec, 60°C for 30sec, 72°C for 30sec, and then 72°C for 2min. The ACTIN2 gene was used as an internal reference control, and its primer sequence was

ACTIN2-F: 5'-GGTAACATTGTGCTCAGTGGTGG-3'

ACTIN2-R: 5'-AACGACCTTAAATCTTCATGCTGC-3'

The expression level was analyzed using the ΔΔCt algorithm.

After PCR identification, 36 p35S::AtZSP1 transgene-positive plants were obtained in T1 generation, and 11 homozygous plants were obtained in T3 generation for subsequent analysis. The results of qRT-PCR showed that the expression level of AtZSP1 gene in homozygous overexpressed plants was significantly higher than that of the non-transgenic wild type, indicating that the expression of AtZSP1 gene in transgenic plants was indeed enhanced ( FIG. 9 ).

For p35S::AtZSP1 overexpression transgenic plants, single-copy plants with a segregation ratio of 3:1 were screened through the T2 generation (according to genetic principles, selfed offspring will produce a segregation ratio of 3:1 after single-copy insertion. Combined with statistics The method of counting the number of resistant seedlings and non-resistant seedlings on the antibiotic medium. The transgenic plant is identified as a single-copy inserted strain by the segregation ratio method, so as to be used for homozygous screening), and p35S is screened out through the T3 generation ::AtZSP1 transgenic homozygous plants, two of which were named as strain p35S::AtZSP1#1 and strain p35S::AtZSP1#2, and were used for the following experiments.

Phenotype observation of embodiment 3atzsp1 mutant and AtZSP1 gene overexpression plant

1. Seed size of mutants and overexpressed plants

The experiment was repeated three times, and 30 seeds were taken from each line for each repetition.

By stereoscopic observation, the seed size of strain atzsp1-1 and strain atzsp1-2 mutants was significantly smaller than that of the wild type, on the contrary, the strain p35S::AtZSP1#1 and strain p35S::AtZSP1 Seeds of #2 were significantly larger than wild type. In order to quantify the degree of seed change, we counted the seed area of each line, and the results showed that the seed area of the mutant line atzsp1-1 and atzsp1-2 was smaller than that of the wild type, and similarly, the strains overexpressing p35S:AtZSP1 The seeds of both line p35S::AtZSP1#1 and line p35S::AtZSP1#2 were larger than wild type. The results are shown in Figure 10. In addition, the 100-seed weight per plant of each strain was counted, and the statistical results are shown in Figure 10. The 100-seed weight of the wild-type seed was 1.70 mg, and the 100-seed weight of the mutant seed was smaller than that of the wild type, only 1.30mg and 1.33mg, decreased by 24% and 22% respectively; while the 100-seed weight of overexpressed plants was significantly higher than that of the wild type, which was 2.73mg and 2.53mg, increased by 38% and 33% respectively. The scale bar in the figure is 1 mm. The SPSS11.5 statistical software was used to process the data, and the experimental results were expressed as mean ± standard deviation, and the t-test test was used. P<0.05 (*) indicated that there was a significant difference compared with wild-type Arabidopsis, P<0.01 (**) means significant difference compared with wild type Arabidopsis, P<0.001 (***) means extremely significant difference compared with wild type Arabidopsis.

2. Mature embryo size of mutants and overexpressed plants

The experiment was repeated three times, and 10-15 mature embryos were taken from each strain for each repetition.

Due to the reduced seed size of the seed mutants, mature embryos of the mutants and overexpression plants were observed and their size was measured areometrically. The results are shown in Figure 11. The mature embryos of the mutant are significantly smaller than those of the wild type. The area of the mature embryo of the wild type is 95.7×10 ³ μm ² , and the area of the mature embryo of the mutant line atzsp1-1 is 71.8×10 ³ μm ^2. The mature embryo area of the mutant line atzsp1-2 is 78.6×10 ³ μm ² , which is 25.0% and 17.9% smaller than that of the wild type, respectively, while the mature embryo area of the overexpression line p35S::AtZSP1#1 The mature embryo area of the overexpression line p35S::AtZSP1# ² was 143.3×10 ³ μm 2 and 162.8×10 ³ μm ² , which were 33.2% and 41.2% higher than that of the wild type, respectively. The scale bar in the figure is 500 μm. The SPSS11.5 statistical software was used to process the data, and the experimental results were expressed as mean ± standard deviation, and the t-test test was used. P<0.05 (*) indicated that there was a significant difference compared with wild-type Arabidopsis, P<0.01 (**) means significant difference compared with wild type Arabidopsis, P<0.001 (***) means extremely significant difference compared with wild type Arabidopsis.

3. Seedling size of mutants and overexpressed plants

The experiment was repeated three times, and 10-15 plant seedlings were taken from each line for each repetition.

Seeds of the five strains were simultaneously sown on MS (Murashige and Skoog basal medium, 0.8% agar powder, pH 5.8) solid medium, treated at 4°C for 3 days, transferred to a 22°C incubator for 8 days, and then transplanted. After being planted in the soil, after 14 days, the above-ground parts of the seedlings were photographed, and the largest leaf area on each seedling of five strains was selected to measure. The results showed that the leaf area of the wild type was 1.01cm ² , and the mutant strain The leaf area of atzsp1-1 and the mutant line atzsp1-2 were 0.62cm ² and 0.63cm ² , which were reduced by 39% and 38% respectively; at the same time, the leaf area of the overexpression plant was significantly larger than that of the wild type, and the overexpression plant The leaf area of the strain p35S::AtZSP1#1 and the overexpression strain p35S::AtZSP1#2 were 1.25cm ² and 1.24cm ² , which increased by 19.2% and 18.5%, respectively. At the same time, the fresh weights of the above-ground parts of these five strains were measured, and the results showed that the fresh weight of the above-ground parts of the wild type was 0.11g, and the fresh weights of the above-ground parts of the mutant strain atzsp1-1 and the mutant strain atzsp1-2 were respectively was 0.068g and 0.063g, decreased by 38.2% and 42.7% respectively, and the fresh weights of the aerial parts of overexpression strain p35S::AtZSP1#1 and overexpression strain p35S::AtZSP1#2 were 0.152g and 0.150g respectively, An increase of 27.6% and 26.7%, respectively (Fig. 12). The scale bar in the figure is 1 cm. The SPSS11.5 statistical software was used to process the data, and the experimental results were expressed as mean ± standard deviation, and the t-test test was used. P<0.05 (*) indicated that there was a significant difference compared with wild-type Arabidopsis, P<0.01 (**) means significant difference compared with wild type Arabidopsis, P<0.001 (***) means extremely significant difference compared with wild type Arabidopsis.

4. Plant height, number of siliques and inflorescence size of mutants and overexpression plants at flowering stage

The experiment was repeated three times, and 25 plants were taken from each line for each repetition.

After the seedlings in the above step 3 were grown in the soil for 28 days, 25 plants were selected for each line, and the photos and plant height statistics were taken. The results were as shown in the figure (Fig. 13). The plant height of the wild type was 26.35cm. The plant heights of the mutant line atzsp1-1 and the mutant line atzsp1-2 were 18.87cm and 19.91cm, which decreased by 28.4% and 24.4% respectively; the overexpression line p35S::AtZSP1#1 and the overexpression line p35S ::The plant height of AtZSP1#2 was higher than that of the wild type, which were 31.02cm and 30.04cm respectively, an increase of 15.1% and 12.3%. The scale bar in the figure is 1 cm. The SPSS11.5 statistical software was used to process the data, and the experimental results were expressed as mean ± standard deviation, and the t-test test was used. P<0.05 (*) indicated that there was a significant difference compared with wild-type Arabidopsis, P<0.01 (**) means significant difference compared with wild type Arabidopsis, P<0.001 (***) means extremely significant difference compared with wild type Arabidopsis.

In addition, the number of siliques and the number of flowers on the main stem of each strain were counted, and the results showed that the number of siliques on the main stem of the wild type was 14.43, and the number of siliques on the main stem of the mutant strain atzsp1-1 and the mutant strain atzsp1 The number of siliques on the main stem of the -2 plant was 7.93 and 8.87, which were reduced by 45.0% and 38.5% respectively; the overexpression line p35S::AtZSP1#1 and the overexpression line p35S::AtZSP1#2 The number of siliques on the main stem of the plants in the wild type was significantly increased, 18.12 and 17.19, respectively, an increase of 20.4% and 16.1%. The statistical results of the number of flowers in the same period showed that the number of flower buds of the wild type was 11.76, and the number of flower buds of the mutant line atzsp1-1 and the mutant line atzsp1-2 were 8.63 and 9.19, respectively, compared with the wild type Respectively decreased by 26.6% and 21.9%; while the number of flower buds of the overexpression line p35S::AtZSP1#1 and the overexpression line p35S::AtZSP1#2 was more than that of the wild type, which were 15.81 and 13.79, respectively. 25.6% and 14.7%. As shown in Figure 13. The scale in the figure is 2mm. The SPSS11.5 statistical software was used to process the data, and the experimental results were expressed as mean ± standard deviation, and the t-test test was used. P<0.05 (*) indicated that there was a significant difference compared with wild-type Arabidopsis, P<0.01 (**) means significant difference compared with wild type Arabidopsis, P<0.001 (***) means extremely significant difference compared with wild type Arabidopsis.

5. Leaf cell size and cell number of mutants and overexpression plants

The experiment was repeated three times, and 10-15 plants were taken from each line for each repetition.

Since the size of the organ is determined by the number of cells and the size of the cells, the cotyledons of the wild type and mutants cultured under light for 6 days were observed. First, the cotyledon area was photographed under a stereoscope. The measurement results showed that the wild type The cotyledon area was 19.31mm ² , and the cotyledon area of the mutant line atzsp1-1 and mutant line atzsp1-2 were 12.16mm ² and 11.10mm ² , respectively. Then observe and measure the palisade cells of the cotyledons under a microscope, the results show (Figure 14), the total number of cells per cotyledon in the wild type is 11.2×10 ³ , the mutant strain atzsp1-1 and the mutant strain atzsp1 The total number of cells in the cotyledon of -2 was 8.5×10 ³ and 7.8×10 ³ , and the number of cells in the cotyledon of the mutant decreased significantly, by 24.1% and 30.3% respectively; the average cell size of the cotyledon was calculated, The results showed that the average cell size of the wild-type cotyledon was 1.75×10 ³ μm ² , while the average cell size of the mutant line atzsp1-1 and the mutant line atzsp1-2 were 1.43×10 ³ μm ² and 1.44× 10 ³ μm ² , decreased by 18.3% and 17.7%, respectively. The above results indicated that AtZSP1 gene may affect leaf size through cell proliferation and cell expansion. The SPSS11.5 statistical software was used to process the data, and the experimental results were expressed as mean ± standard deviation, and the t-test test was used. P<0.05 (*) indicated that there was a significant difference compared with wild-type Arabidopsis, P<0.01 (**) means significant difference compared with wild type Arabidopsis, P<0.001 (***) means extremely significant difference compared with wild type Arabidopsis.

The present invention classifies unknown small peptide proteins of Arabidopsis thaliana, and when identifying the single-copy gene whose amino acid length is less than 100, a gene encoding 57 amino acids is found, and the gene is named AtZSP1. The T-DNA insertion mutant of this gene was identified, and another homozygous mutant was obtained using CRISPR/Cas9 technology, and both mutants were complete knockout mutants. The phenotype of the mutant was observed, and the results showed that the seed size and 100-grain weight of the mutant were significantly smaller than that of the wild type, and the plants were short, and the size of leaves, the number of flower buds and the number of siliques on the main stem were all less than those of the wild type. It shows that the deletion of AtZSP1 gene affects the seed and organ size of Arabidopsis. In addition, the phenotype of the obtained homozygous overexpression plants was observed, and it was found that the phenotype of the overexpression plants was completely opposite to that of the mutant. Since the size of plant organs is determined by the number of cells and the size of the cells, the number and size of the cotyledon cells of the wild type and mutants were counted. The average cell size of the cotyledons was also smaller than that of the wild type. It shows that AtZSP1 gene may affect the size of organs by controlling cell proliferation and cell expansion. In addition, the polypeptide encoded by this gene has homologous proteins in rapeseed, corn, rice, Chinese cabbage, soybean, millet, and sorghum, and the sequences of these proteins are relatively conserved in these species, suggesting that the AtZSP1 gene may be found in crop varieties. There are good prospects for improvement.

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

<110> China Agricultural University

<120> Small peptides regulating the size of plant seeds and organs, their coding genes and applications

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 57

<212> PRT

<213> Arabidopsis thaliana

<400> 1

Met Gln Lys Ser Phe Ser Leu Ile Gln Thr Val Ala Ile Ser Gly Val

1 5 10 15

Phe Ser Ala Val Ser Cys Trp Tyr Gly Phe Met Phe Gly Arg Glu Ser

20 25 30

Ala Arg Lys Glu Leu Gly Gly Leu Ile Glu Glu Leu Arg Arg Gly Gly

35 40 45

Ser Asn Ser Asp Ser Thr Pro His Ser

50 55

<210> 2

<211> 174

<212>DNA

<213> Arabidopsis thaliana

<400> 2

atgcaaaaat cgttctcgtt gatccaaaca gtggcaatct ccggcgtatt ctccgccgtc 60

tcatgctggt atggattcat gttcggtaga gaatcagcga gaaaagagct cggaggtttg 120

atcgaagagc tccgtcgtgg aggctccaat tctgattcca ctccccattc ctga 174

<210> 3

<211> 1948

<212>DNA

<213> Arabidopsis thaliana

<400> 3

aaacgggcat cggtatgaag gagcctggca tgaggggaga aggcaaggac ttggtatgta 60

cacgttcagg aatggtgaga cacaggcagg ccattgggaa gacggagttc ttaactgtcc 120

taccgagcag accactcgcc ctgattcatc gttttccatc agtcattcca aggttgtcga 180

cactgtccag gtaccttata atttatctta ctgctgctca tatatattaa gttagagttt 240

agatctttaa gaaattatct tactgctgcc aatgattgag tacccacatg agattgatc 300

ctgctccctc tctctctttg atcaacagca agcaaggaaa gcagccaaga aagcacgcga 360

agttgtgaaa gttgaagaga gagtgaacag agcagtgatg gtcgcaaacc gagcagcaaa 420

tgctgctaga gttgctgcta caaaggctgt ccaaacccaa aatttcatt gtagtagtgg 480

tgacgatccc ttgtgagttc ctgcggcaaa gattttgaca taatcgcagt catgacctta 540

cctcgaactt ctacgaagaac agaagagtga aagagagatg tttagagaac agcgtcagaa 600

ttcgcaatct ccgttgagat tttcttgaag gtgaaccact tggaaccaat gtcgagatct 660

cataagtaat aagtaagaga tctggctatg gagcaagtat tacaggttta ctttttcttt 720

tctcccactt gttttatgag caggctggtt taaaactgta catatgttgt tgaagagtaa 780

gcaatgtgtg ttaacaaaag atctttgtaa caagcgtaac ccatgtaaga gaagtaacaa 840

aacattgttt tacttttaca acgtgttgaa aataacacct tttttttact agatttctat 900

ggatagaaat tagaaaatga catgagaatt ccagattcaa atcaacttga caaaaccaac 960

aagggaaaga gtaaagatat ctggtctgtg acaaggtcca agtagagacg aattacagat 1020

catctaggta actgtgctta tggttcttca aaccctttca gcaaatccta gcagccagcc 1080

tttgtgtttg attaaagtgg gctgggctaa aatattggcc gaggcccatc aagtgattct 1140

ctcccgctca agtcttttaa tccttgagga gaaaatggac tggaaaattc acggaacttt 1200

aacagaaaga tgcaaaaatc gttctcgttg atccaaacag tggcaatctc cggcgtattc 1260

tccgccgtct catgctggtg agtgattgtt cccgtctttc aagctcttct tcttcaattc 1320

tcgtcttctt ctctttccat gtctcatctt tagggtttaa gctgccttac tcaatttcca 1380

ggtatggatt catgttcggt agagaatcag cgagaaaaga gctcggaggt ttgatcgaag 1440

agctccgtcg tggaggctcc aattctgatt ccactcccca ttcctgagtt aggcattcca 1500

atttcccagg acggaggagg ttcttcgtta tccgttgcta agttagtttc cgatttcgat 1560

taatgttcat gaaaattgat caattgttgt ataaaccgat gtgtttgaca aatcctttga 1620

gttaattcaa gtatgaaaga ttatcgtttg aacgagagat gcatattgat tgatgataag 1680

agaaactcac attgattgag ataatataaa actgtaggag aaaaatgcat agtatttgag 1740

attaagagta tgaagatgat gtacaaggaa aactcatagg aaacatctaa gtgtacatca 1800

tcttgatttt gcttctcttt gctctgactc tgacccagaa ttgtgcaaat ctatagcttt 1860

aacactctta ctggtttgat ctctctagtt atttatcaga caggaataca ataaacacca 1920

cactatatgt atgtacctat gtgggaaa 1948

<210> 4

<211> 660

<212>DNA

<213> Arabidopsis thaliana

<400> 4

aaattcacgg aactttaaca gaaagatgca aaaatcgttc tcgttgatcc aaacagtggc 60

aatctccggc gtattctccg ccgtctcatg ctggtatgga ttcatgttcg gtagagaatc 120

agcgagaaaa gagctcggag gtttgatcga agagctccgt cgtggaggct ccaattctga 180

ttccactccc cattcctgag ttaggcattc caatttccca ggacggagga ggttcttcgt 240

tatccgttgc taagttagtt tccgatttcg attaatgttc atgaaaattg atcaattgtt 300

gtataaaccg atgtgtttga caaatccttt gagttaattc aagtatgaaa gattatcgtt 360

tgaacgagag atgcatattg attgatgata agagaaactc aattgattg agataatata 420

aaactgtagg agaaaaatgc atagtatttg agattaagag tatgaagatg atgtacaagg 480

aaaactcata ggaaacatct aagtgtacat catcttgatt ttgcttctct ttgctctgac 540

tctgacccag aattgtgcaa atctatagct ttaacactct tactggtttg atctctctag 600

ttattattca gacaggaata caataaacac cacactatat gtatgtacct atgtgggaaa 660

<210> 5

<211>626

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

atatatggtc tcgattggcc ggagattgcc actgttgttt tagagctaga aatagcaagt 60

taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttt 120

gcaaaatttt ccagatcgat ttcttcttcc tctgttcttc ggcgttcaat ttctggggtt 180

ttctcttcgt tttctgtaac tgaaacctaa aatttgacct aaaaaaaatc tcaaataata 240

tgattcagtg gttttgtact tttcagttag ttgagttttg cagttccgat gagataaacc 300

aatattaatc caaactactg cagcctgaca gacaaatgag gatgcaaaca attttaaagt 360

ttatctaacg ctagctgttt tgtttcttct ctctggtgca ccaacgacgg cgttttctca 420

atcataaaga ggcttgtttt acttaaggcc aataatgttg atggatcgaa agaagagggc 480

ttttaataaa cgagcccgtt taagctgtaa acgatgtcaa aaacatccca catcgttcag 540

ttgaaaatag aagctctgtt tatatattgg tagagtcgac taagagattg actcaccagc 600

atgagacggg tttagagacc aataat 626

<210> 6

<211> 250

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

atgcaaaaat cgttctcgtt gatccaaaca tctcatgctg gtgagtgatt gttcccgtct 60

ttcaagctct tcttcttcaa ttctcgtctt cttctctttc catgtctcat ctttagggtt 120

taagctgcct tactcaattt ccaggtatgg attcatgttc ggtagagaat cagcgagaaa 180

agagctcgga ggtttgatcg aagagctccg tcgtggaggc tccaattctg attccactcc 240

ccattcctga 250

<210> 7

<211> 146

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

atgcaaaaat cgttctcgtt gatccaaaca tctcatgctg gtatggattc atgttcggta 60

gagaatcagc gagaaaagag ctcggaggtt tgatcgaaga gctccgtcgt ggaggctcca 120

attctgattc cactccccat tcctga 146

<210> 8

<211> 30

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 8

Met Gln Lys Ser Phe Ser Leu Ile Gln Thr Ser His Ala Gly Met Asp

1 5 10 15

Ser Cys Ser Val Glu Asn Gln Arg Glu Lys Ser Ser Glu Val

20 25 30

Claims (10)

1. The application of polypeptide, it is characterized in that, described application is following any one: D1) The application of polypeptides in regulating the size of plant seeds and/or organs; D2) The application of polypeptides in the preparation of products for regulating the size of plant seeds and/or organs; D3) Use of polypeptides for the cultivation of plants with increased or decreased seed and/or organ size; D4) Use of polypeptides for the manufacture of products for the cultivation of plants with increased or decreased seed and/or organ size; D5) Application of polypeptides in plant breeding; The polypeptide is any of the following: A1) The amino acid sequence is the polypeptide of SEQ ID No.1; A2) A fusion polypeptide obtained by linking tags at the N-terminal and/or C-terminal of A1); A3) The amino acid sequence is the polypeptide of SEQ ID No.8; A4) A fusion polypeptide obtained by linking a tag at the N-terminal and/or C-terminal of A3). 2. The application of biological materials, characterized in that the application is any one of the following: E1) Use in regulating plant seed and/or organ size; E2) Application in the preparation of products regulating the size of plant seeds and/or organs; E3) Use in the cultivation of plants with increased or decreased seed and/or organ size; E4) Use in the manufacture of products for the cultivation of plants with seeds and/or organs of increased or decreased size; E5) Application in plant breeding; The biological material is any of the following: B1) a nucleic acid molecule encoding the polypeptide described in 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); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2); B6) Transgenic plant tissue containing the nucleic acid molecule described in B1), or a transgenic plant tissue containing the expression cassette described in B2); B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassettes described in B2); D1) A nucleic acid molecule that inhibits or reduces the expression of the polypeptide-encoding gene described in claim 1; D2) an expression cassette containing the nucleic acid molecule of D1); D3) A recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2); D4) A recombinant microorganism containing the nucleic acid molecule described in D1), or a recombinant microorganism containing an expression cassette described in D2), or a recombinant microorganism containing a recombinant vector described in D3); D5) A transgenic plant cell line containing the nucleic acid molecule described in D1), or a transgenic plant cell line containing the expression cassette described in D2); D6) Transgenic plant tissues containing the nucleic acid molecules described in D1), or transgenic plant tissues containing the expression cassettes described in D2); D7) A transgenic plant organ containing the nucleic acid molecule described in D1), or a transgenic plant organ containing the expression cassette described in D2). 3. The application according to claim 2, characterized in that, the nucleic acid molecule in B1) is a DNA molecule as shown in b1) or b2) as follows: b1) The coding sequence (CDS) is the DNA molecule or cDNA molecule shown in SEQ ID No.2; b2) The nucleotide sequence is the DNA molecule or cDNA molecule shown in SEQ ID No.2. 4. A method for increasing the size of plant seeds and/or organs, characterized in that the method comprises increasing the expression and/or activity of the polypeptide described in claim 1 in plants to increase the size of plant seeds and/or organs size. 5. The method according to claim 4, characterized in that said improving or increasing the expression and/or activity of the polypeptide of claim 1 in the plant is achieved by increasing the expression level of the gene encoding the polypeptide in the plant. 6. A method for reducing the size of plant seeds and/or organs, characterized in that, the method comprises inhibiting or reducing the expression of the polypeptide encoding gene described in claim 1 and/or the polypeptide described in claim 1 in the plant activity to reduce plant seed and/or organ size. 7. The method according to claim 6, characterized in that, the method for inhibiting or reducing the expression of the polypeptide-encoding gene described in claim 1 in the plant and/or the activity of the polypeptide described in claim 1 is to The 31st-58th nucleotides of SEQ ID No.2 in the plant genome are deleted. 8. The application according to any one of claims 1-3 or the method according to any one of claims 4-7, wherein the plant is any one of the following: F1) monocot or dicot; F2) Plants of the order Lilyaceae, Grasses or Beans; F3) Brassicaceae, grasses or legumes; F4) Arabidopsis, Brassica, Zea, Oryza, Setaria, Sorghum or Glycine; F5) Arabidopsis, rapeseed, Chinese cabbage, corn, rice, millet, sorghum or soybean. 9. Use of the method of any one of claims 4-7 for the cultivation of plants having increased or decreased seed and/or organ size. 10. Use of the method according to any one of claims 4-7 in plant breeding.

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