CN112695042A - ZmD53 application of gene in regulating development of maize tassel branches or breeding new variety of density-resistant plants - Google Patents

Abstract

The invention discloses an application of ZmD53 gene in regulating and controlling development of maize tassel branches or breeding a new variety of close planting resistant maize. Firstly, a corn mutant of ZmD53 gene is constructed, and the mutation of ZmD53 gene is found through phenotype observation to cause the obvious reduction of the number of corn tassel branches; the invention further discovers that ZmD53 is an important factor in regulating and controlling a signal path of the corn strigolactone through molecular mechanism research, can regulate and control the development or the branch number of the corn tassel through regulating and controlling the expression of ZmD53 genes in the corn, and has application prospects in the aspects of cultivating novel varieties of ideal plant type corn which is resistant to close planting and suitable for mechanized harvesting and the like.

Description

ZmD53 application of gene in regulating development of maize tassel branches or breeding new variety of density-resistant plants

Technical Field

The invention relates to a new application of ZmD53 gene, in particular to an application of ZmD53 gene in regulating and controlling development of maize tassel branches or breeding a new variety of dense planting resistance, belonging to the field of new application of ZmD53 gene.

Background

Corn is not only an important grain crop, but also an important raw material in the industries of food, chemical industry, fuel, medicine and the like. At present, corn is the first crop with the widest planting area and the highest yield per unit in China. However, due to population growth, dietary structure change, urbanization and increase of energy supply pressure, the improvement of the unit yield of corn has become a key technical measure for guaranteeing the food safety in China.

A large number of researches show that the improvement of the density tolerance and the planting density of the variety is the key for improving the yield per unit of the corn. As a large country for corn planting and production, the planting density of corn in the United states has increased from-30000 plants/hectare in the thirty years of the last century (Cardwell,1982) to-70000 plants/hectare in the present era (62000-104000 plants/hectare) (Mansfield and Mumm, 2014); at the same time, the yield per unit is also increased from 1287 kg/hectare to 9595 kg/hectare (statistics in 2010) (Mansfield and Mumm, 2014; USDA-NASS, 2012).

In the aspect of corn breeding, proper plant height and ear position, proper ear branch number, optimal growth period and optimal leaf angle are key indexes for obtaining ideal plant type, yield and biomass, and are also the basis for cultivating new varieties suitable for mechanized operations. Tassel size (tassel branch number and length) is an important target trait for corn domestication and genetic improvement. The over-developed tassels affect the canopy structure of the plant type and reduce the overall light energy utilization rate of the plant. However, tassel branch development, which begins at the reproductive growth stage, is essentially the development of the lateral meristem of the plant. How to regulate and control the tassel branch and how to regulate and control the operation of a path, how to utilize the tassel branch, and how to contribute theoretical basis and gene resources for cultivating a new variety with excellent high efficiency and density resistance. The maize tassel branch number is a typical quantitative trait, and the differentiation and development process is synergistically regulated and controlled by plant hormones, environment and genetic factors. A number of pathways have been identified to be involved in regulating maize tassel development, including the CLV/WUS pathway, the auxin pathway, the RAMOSA pathway, the SPL pathway, and others (Justine et al, 1999; Taguchi-Shiobara et al, 2001; Gallavtti et al, 2004,2010; Vollbrecht et al, 2005; Bommert et al, 2005; Skirpan et al, 2009; Chuck et al, 2014).

Research has shown that the increase of the American corn yield per plant and heterosis is not obvious in the breeding process of decades, and the increase of the corn yield per plant is more due to the continuous increase of the planting density and the variety tightness (Duvick, 2005; Duvick and Cassman, 1999; Duvick, 1997; Hammer et al, 2009). Research on Chinese maize varieties in different years also shows that the near modern varieties all show more adaptability to high-density cultivation conditions in terms of important agronomic traits such as yield, photosynthetic efficiency, lodging resistance, stalk-empty rate, etc., compared to the early varieties (Baizhuang et al, 2015; Likefeng et al, 2013; Liuxin et al, 2012; Qianchunong et al, 2012; Tangbin et al, 2013). Therefore, the improvement of the variety density and planting density is an important target and trend of modern corn breeding and production. In recent years, along with the development of society, the labor cost in China is higher and higher, and agriculture is also developed towards mechanization and intensification; under these pressures, the cultivation of close-planting tolerant, mechanically harvested, ideal plant-type maize varieties is an important outlet for increasing maize yield per unit and increasing grower profits (Gong et al, 2015).

Therefore, the identification of the key genes for regulating the tassel branch number and the tassel size and the analysis of the genetic regulation network and the molecular mechanism of the key genes have guiding significance for the genetic improvement of the tassels.

Disclosure of Invention

One of the purposes of the invention is to identify key genes for regulating the tassel branch number or the tassel size;

the second purpose of the invention is to apply the identified key genes for regulating the tassel branch number and the tassel size to the genetic improvement of the tassels of the corn or culture a new variety of the corn with close planting resistance;

the above object of the present invention is achieved by the following technical solutions:

the invention firstly constructs a corn dominant mutant material ZmD53 driven by ZmD53 endogenous promoter_promoter:Zmd53^(RGKRAT)(zmd53, Rice D53 mutation was simulated, and 18 bases were deleted by deleting six amino acids 819^RGKRAT824, which results in ZmD53 not being able to be degraded by the ubiquitination pathway); a plurality of independent transgenic lines all show a defect phenotype with reduced tassel branch number; this shows that ZmD53 is an important factor in the regulation of the strigolactone signal pathway in maize, and plays an important role in the regulation of the strigolactone signal pathway in maize and the agronomic traits of maize.

To elucidate the molecular mechanism of ZmD53 gene regulation of tassel branch number, the invention further finds that the strigolactone analogue GR24 can degrade ZmD53 protein, but the mutated Zmd53 protein does not respond to GR 24), and the test result shows that ZmD53 is a member of the strigolactone signaling pathway in maize. On the basis, the molecular mechanism of regulating the tassel branch number is further analyzed, and the result shows that ZmD53 can regulate the downstream gene expression through the interaction with SPL proteins UB3 and TSH4, and the result suggests that the SL signal pathway and the SPL signal pathway in the corn interact to regulate the development process of the tassel branches of the corn synergistically.

Thus, the present invention provides a method for reducing the number of tassel branches in corn or for reducing the size of tassels in corn, comprising:

constructing a knock-out vector of the ZmD53 gene; and transforming the constructed gene knockout vector into a corn plant, knocking out or mutating an ZmD53 gene in the corn, and reducing the number of tassel branches or reducing the tassels in the obtained transgenic plant.

The invention also provides a method for cultivating a new variety of close planting resistant corn, which comprises the following steps: constructing a knock-out vector of the ZmD53 gene; and transforming the constructed gene knockout vector into a corn plant, and screening to obtain a new corn variety with reduced tassel branches or reduced tassels.

The skilled person can mutate or knock out ZmD53 gene in corn by using conventional methods such as conventional gene knock-out or gene editing techniques, for example, constructing ZmD53 gene knock-out vector or constructing CRISPR/Cas9 gene editing vector by using gene editing techniques, etc., and can mutate or knock out ZmD53 gene in corn, which are all well known to those skilled in the art.

The nucleotide sequence of the ZmD53 gene is shown in SEQ ID No. 1.

The invention further provides a method for increasing the number of tassel branches of corn or enlarging the tassel, which comprises the following steps:

overexpression is carried out on the ZmD53 gene in plants to obtain transgenic plants; such as: the ZmD53 gene is operably connected with an expression regulatory element to obtain a recombinant plant expression vector for expressing the gene in corn; transforming corn with the recombinant plant expression vector to over-express ZmD53 gene in corn plant.

The invention discloses a recombinant expression vector containing the ZmD53 gene and a recombinant host cell containing the recombinant expression vector; comprises that the ZmD53 gene is operably connected with an expression regulatory element to obtain a recombinant plant expression vector; the recombinant plant expression vector can consist of a 5 'end non-coding region, ZmD53 gene and a 3' non-coding region; wherein, the 5' non-coding region can comprise a promoter sequence, an enhancer sequence or/and a translation enhancing sequence; the promoter can be a constitutive promoter, an inducible promoter, a tissue or organ specific promoter; the 3' non-coding region may comprise a terminator sequence, an mRNA cleavage sequence, and the like. Suitable terminator sequences can be taken from the Ti-plasmid of Agrobacterium tumefaciens, for example the octopine synthase and nopaline synthase termination regions.

The recombinant plant expression vector may also contain a selectable marker gene for selection of transformed cells or tissues. The marker gene includes: genes encoding antibiotic resistance, genes conferring resistance to herbicidal compounds, and the like. In addition, the marker gene also comprises phenotypic markers, such as beta-galactosidase, fluorescent protein and the like.

The transformation protocol, and the protocol for introducing the polynucleotide or polypeptide into a plant, may vary depending on the type of plant (monocot or dicot) or plant cell used for transformation. Suitable methods for introducing the polynucleotide into a plant cell include: microinjection, electroporation, agrobacterium-mediated transformation, direct gene transfer, and high-speed ballistic bombardment, among others. In particular embodiments, the encoding gene may be provided to the plant using a variety of transient transformation methods. The transformed cells can be regenerated into stably transformed plants using conventional methods (McCormick et al plant Cell reports.1986.5: 81-84).

The target plants include but are not limited to: a monocotyledonous plant or a dicotyledonous plant. More preferably, the target plant is maize.

The invention identifies a key gene ZmD53 for regulating the tassel branch number in corn, further finds that ZmD53 is an important factor for regulating a strigolactone signal channel of corn, and regulates the tassel branch number of corn by regulating the expression of ZmD 53; the invention further analyzes the genetic control network and the molecular mechanism; the expression of ZmD53 is regulated to regulate the tassel branch number, modify the corn plant type or culture a new high-yield variety with close planting resistance.

Definitions of terms to which the invention relates

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, which include PNAs (peptide nucleic acids), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, etc.). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly specified. In particular, degenerate codon substitutions may be achieved by generating sequences in which the 3 rd position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res.19:5081 (1991); Ohtsuka et al, J.biol.chem.260: 2605-S2608 (1985); and Cassol et al (1992); Rossolini et al, Mol cell. probes 8:91-98 (1994)).

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to mean a polymer of amino acid residues. That is, the description for a polypeptide applies equally to the description of a peptide and to the description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are a non-naturally encoded amino acid. As used herein, the term encompasses amino acid chains of any length, including full-length proteins (i.e., antigens), in which the amino acid residues are linked via covalent peptide bonds.

The term "recombinant host cell strain" or "host cell" means a cell comprising a polynucleotide of the present invention, regardless of the method used for insertion to produce the recombinant host cell, e.g., direct uptake, transduction, f-pairing, or other methods known in the art. The exogenous polynucleotide may remain as a non-integrating vector, such as a plasmid, or may integrate into the host genome. The host cell may be a prokaryotic cell or a eukaryotic cell, and the host cell may also be a monocotyledonous or dicotyledonous plant cell.

The term "operably linked" refers to a functional linkage between two or more elements that may be operably linked and may or may not be contiguous.

The term "recombinant plant expression vector" means one or more DNA vectors used to effect plant transformation; these vectors are often referred to in the art as binary vectors. Binary vectors, together with vectors with helper plasmids, are most commonly used for agrobacterium-mediated transformation. Binary vectors typically include: cis-acting sequences required for T-DNA transfer, selectable markers engineered to be capable of expression in plant cells, heterologous DNA sequences to be transcribed, and the like.

The term "transformation" refers to a process of introducing a heterologous DNA sequence into a host cell or organism.

The term "expression" refers to the transcription and/or translation of an endogenous gene or transgene in a plant cell.

Drawings

FIG. 1 shows the change in expression level of 1 ZmD53 gene in each tissue of maize.

FIG. 2 phenotypic analysis of mutant plants; A.ZmD53: vector construction map of Zmd 53; B. the number of tassel branches of the two lines of ZmD53 was significantly reduced compared to wild-type WT; c, a scanning electron microscope imaging picture of the tassel primordium of WT; d, a scanning electron microscope imaging picture of the tassel primordium of ZmD53; E. and counting the number of tassel branches of the two lines of WT and ZmD53 mutant.

FIG. 3 ZmD53 and Zmd53 response to GR 24; both ZmD53(a) and Zmd53(C) are nuclear localization proteins in maize protoplasts, ZmD53 respond to the strigolactone analogue GR24, being able to be degraded by it (B), but Zmd53 does not respond to GR24 (D).

Fig. 4 ZmD53 interacts with UB3 and TSH4 in vitro.

Detailed Description

The invention is further described below in conjunction with specific embodiments, the advantages and features of which will become apparent from the description. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.

Test example 1 construction of maize ZmD53 mutant and phenotypic Observation test

Construction of ZmD53 dominant mutant material ZmD53 driven by endogenous promoter_promoter:Zmd53^(RGKRAT)(zmd53, Rice D53 mutation was simulated, and 18 bases were deleted by deleting six amino acids 819^RGKRAT824, which results in ZmD53 not being able to be degraded by the ubiquitination pathway).

The specific construction method comprises the following steps:

the CPB vector is used as a framework, the amplified D53 promoter is connected into the framework by an infusion method, the D53cdna is divided into two sections by taking a mutation region as a boundary, the middle 18 bases are deleted by the infusion method, and the two sections are fused into the CPB vector framework connected with the D53 promoter.

The primer sequences used were as follows:

D53promoter-F：TACGTAGGGTGGTGGTGCACGT；

D53promoter-R：TTAACGGCAAGCCAGAGGGTG；

D53-F:TTTCTTTCCCGTTTCACTCCT；

D53-R:CCAATCCAGAATTATTCTCGAGG；

the transformation method comprises the following steps: transforming maize embryo with EHA105 agrobacterium mediation.

Transgenic plants were obtained via basta resistance screen.

It was observed that multiple independent transgenic lines all exhibited a defective phenotype with reduced tassel branch number (fig. 1 and 2).

Test example 2 molecular mechanism test of regulating tassel branch number by 2 ZmD53 Gene

In this experiment ZmD53 and a mutant form of Zmd53 were constructed into the subcellular localization expression vector 1305, maize protoplasts were transformed to obtain a transient expression system, and after treatment with 5. mu.MGR 24 in the treated group for 4h, ZmD53 and Zmd53 expression patterns were observed together with the control group. Strigolactone analogue GR24 was found to be able to degrade the ZmD53 protein, but the mutated Zmd53 protein was not responsive to GR24 (fig. 3), indicating that ZmD53 is a member of the strigolactone signaling pathway in maize.

Furthermore, the molecular mechanism of regulating the tassel branch number is analyzed, and the experiment explores the correlation between ZmD53 and SPL protein by a yeast two-hybrid method. ZmD53 and SPL proteins UB2, UB3 and TSH4 are respectively constructed on two vectors, namely PGADT7 and PGBKT7, and the mutual relation of the two vectors is verified by cotransformation of yeast strain AH 109; it was found that ZmD53 can co-regulate downstream gene expression by interacting with SPL proteins UB3 and TSH4 (FIG. 4), and this result suggests that the interaction of SL signaling pathway and SPL signaling pathway in maize synergistically regulates maize tassel branch development process.

Sequence listing

<110> southern China university of agriculture

Application of <120> ZmD53 gene in regulation of development of maize tassel branches or cultivation of new variety resistant to close planting

<130> GD-2001-190828A

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

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<212> DNA

<213> Zea mays L

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atcgcctcgc tcctcgcccc caccgccgcg ccgctgctcc gcgacgcgct cgcccgcgcc 180

cgcagcgccg cctactcccc gcgcctccag ctcaaggcgc tcgacctctg cttcgccgtc 240

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tccgcctccg ccagcaccag caatgacccc cagcagcacg agccccccgt cgccaactcc 360

ctcatggccg ccatcaagcg ctcgcaggcc aaccagcgcc ggaacccgga cacgttccac 420

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cttgtcctcg ccatcctcga cgacccgctc gtcagccgcg tcttcgccga cgcgggattc 540

cgcagcaacg agatcaaggt cgccatcctc cgccccgcgc cgcccgtccc gctgctcggc 600

cgcctcccca cgcgcgcgcg cccgccgccg ctcttcctct gcagctttgc cgccgcggac 660

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tgccgccgca tcaccgacat cctcgcccgc ggacgcaacc ccatgctcgt cggcgtcggg 780

gccgcctccg cagccgcaga tttcgccaag gcgtccccgt accgcatcgt ccccatcggc 840

cccgcctctg ccaataatca ggcggcaccg acgacgccca cctctggcct tatcttcagc 900

atcggtgatc tcaaggacct ggtgcccgac gaggccgacc tgcaggacgc ggctcgccgg 960

gtggtggcgg aggtcacgcg cctgctcgag acgcacagac ctgctgcccg ccaaacccaa 1020

acggtctggg tcatgggctg gtccgccacc tacgagacct acctcgcctt cctttccaag 1080

ttcccactcg tcgacaagga ctgggaactc cagctgctgc cgatcaccgc cgtgcgcgac 1140

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ttgtccatgc ctgccactac aagcttcgcg gagtcatttg ttccttttgg aggtcttttg 1260

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caacattgca acgatagata tgagcaagaa gttgcctcta tcattagagg aagtggcgtt 1380

acagctgatg ctcaccaaga aggtctacct tctctgttgc agaatggcgg catgatgggt 1440

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Claims (10)

1. The application of ZmD53 gene in the regulation of maize tassel branch development. 2. according to the application described in claim 1, it is characterized in that, described regulation and control corn tassel branch development is to reduce the number of corn tassel branches or retard corn tassel development. 3. according to the described application of claim 1, it is characterized in that, described regulation and control corn tassel branch development is to make tassel smaller. 4. according to the described application of claim 1, it is characterized in that, described regulation and control corn tassel branch development is to increase the number of corn tassel branches. 5. according to the described application of claim 1, it is characterized in that, described regulation and control corn tassel branch development is to make corn tassel bigger. 6. A method for reducing the number of branches of maize tassel or making maize tassel smaller, comprising: constructing a knockout vector or gene editing vector of the ZmD53 gene; editing the constructed gene knockout vector or gene editing The vector is transformed into maize, the ZmD53 gene in maize is knocked out or mutated, and the number of tassel branches in the obtained transgenic plants is reduced. 7. A method for cultivating a high-yield corn variety resistant to dense planting, comprising: constructing a knockout vector or a gene editing vector of the ZmD53 gene; transforming the constructed gene knockout vector or gene editing vector into a corn plant, and converting the ZmD53 gene was knocked out or mutated, and new maize varieties with reduced tassel branches were obtained by screening. 8. A method for increasing the number of branches of corn tassel or making corn tassel larger, comprising: overexpressing the ZmD53 gene in a plant to obtain a transgenic plant. 9. according to the method described in claim 8, it is characterized in that, comprising: ZmD53 gene is operably connected with expression control element, obtains the recombinant plant expression vector expressing ZmD53 gene in plant; Described recombinant plant expression vector Transform maize to overexpress or overexpress the ZmD53 gene in maize. 10. The method according to any one of claims 1-9, wherein the nucleotide sequence of the ZmD53 gene is shown in SEQ ID No.1.

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