CN115466747A

CN115466747A - Glycosyltransferase ZmKOB1 gene and application thereof in regulating and controlling maize ear fructification character or development

Info

Publication number: CN115466747A
Application number: CN202110653667.1A
Authority: CN
Inventors: 王海洋; 陈家欢; 李耀耀; 赵永平; 赵斌斌; 王宝宝; 孔德鑫
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2021-06-11
Filing date: 2021-06-11
Publication date: 2022-12-13
Anticipated expiration: 2041-06-11
Also published as: CN115466747B

Abstract

The invention discloses a glycosyltransferase ZmKOB1 gene and application thereof in regulating and controlling the fructification character or development of corn ears. According to the invention, glycosyltransferase ZmKOB1 gene is separated from corn, and a ZmKOB1 overexpression strain with plant phenotype seed number, ear length and ear weight increased is obtained by using a gene overexpression technology; further use of the geneKnocking out a ZmKOB1 gene of the corn by an editing technology to obtain a mutant with a phenotype of reduced kernel number, shortened ear length and reduced ear weight; hybridizing with ZmKOB1 super-expression strain under the genetic background of maize inbred line to obtain F ₁ And finally, determining that the ZmKOB1 is used for genetic improvement of the row grain number, the ear length and the ear grain weight of the hybrid, thereby improving the grain yield of the hybrid. The invention provides theoretical basis for development genetic improvement of corn row grain number, ear length, ear grain weight, heterosis utilization and the like, and has application prospect in breeding new corn varieties or genetic improvement of corn.

Description

Glycosyltransferase ZmKOB1 gene and application thereof in regulating and controlling maize ear fructification character or development

Technical Field

The invention relates to a glycosyltransferase ZmKOB1 gene and application thereof, in particular to a glycosyltransferase ZmKOB1 gene separated from corn and application thereof in regulating and controlling the corn ear setting or development traits and cultivating a high-yield and new corn variety, belonging to the field of glycosyltransferase ZmKOB1 genes and application thereof.

Background

Corn (Zea mays L.) is one of three major food crops in the world, is also the first major crop in China, and plays an important role in national economy. With the increasing population, the cultivated land area of China is limited, and the corn planting area is reduced by the adjustment of the national industrial structure recently, so that the corn with the functions of grains, feeds, industrial raw materials and the like faces greater demands under the environment of increasing economy and increasingly tense energy sources, and under the circumstance, the improvement of the yield of a single corn plant has great strategic significance for guaranteeing the grain safety of China. The yield per unit of the corn mainly comprises the weight of hundred grains, the number of grains in a row, the number of rows of ears, the number of effective ears in a unit area and the like, wherein the number of the grains in the row is used as an important component factor of the yield of the corn, the heritability of the corn is high, and the corn is in positive correlation with the yield. Therefore, the method has important theoretical significance and application value in digging genes influencing the corn row grain number and analyzing the regulation mechanism of the genes.

Plant Glycosyltransferases (Glycosyltransferases) are a class of enzymes that can transfer a sugar group to a small molecule, including plant hormones, polypeptides, proteins, and secondary metabolites. The existing research results show that glycosyltransferase participates in the response to abiotic stress and influences the growth and development processes of plant hypocotyl elongation, leaf development, seed germination, grain size, sex determination and the like by regulating and controlling the dynamic balance of auxin in a plant body.

A large number of previous research results show that the QTL for controlling the corn kernel number is large in number, but the genetic effect of the QTL sites is small, and the QTL sites for controlling the corn kernel number contained in parents of different groups located by researchers are not consistent, which indicates that the control of the corn kernel number is a complex quantitative trait controlled by a series of micro-effective polygenes. Although there are many QTL sites reported to regulate maize grain number, there are still few genes that have been successfully cloned. In addition, some researchers have developed the cloning and research of genes using maize ear mutants; it has been found that ear mutants are often caused by defects in hormone synthesis or signal transduction pathways. However, the reported maize mutants have too extreme phenotypes, which have great negative effects on maize plant type, biomass and yield, and are difficult to be applied in actual production. Therefore, it is urgently needed to find key genes for regulating and controlling maize ear traits, which can be applied to breeding.

Disclosure of Invention

One of the purposes of the present invention is to isolate the glycosyltransferase ZmKOB1 gene from maize;

the other purpose of the invention is to apply the identified key gene (glycosyltransferase ZmKOB 1) for regulating and controlling the ear traits of the corn, the utilization of heterosis and the like to genetic improvement of the ear of the corn or to breeding of the corn, including the cultivation of a corn inbred line and hybrid seeds.

In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:

the ZmKOB1 gene is firstly separated from corn, and the polynucleotide sequence of CDS is shown in (a), (b), (c), (d) or (e):

(a) The polynucleotide shown in SEQ ID No. 1; or

(b) Polynucleotide for coding amino acid shown in SEQ ID No. 2; or

(c) A polynucleotide capable of hybridizing under stringent hybridization conditions to the complement of the polynucleotide of SEQ ID No. 1; or

(d) A polynucleotide having at least 90% or more homology with the polynucleotide represented by SEQ ID No. 1; or

(e) And a polynucleotide variant having deletion, substitution or insertion of one or more bases based on the polynucleotide represented by SEQ ID NO.1, wherein the polynucleotide variant encodes a protein having the function or activity of a glycosyltransferase.

In addition, the polynucleotide shown in SEQ ID No.1 can be optimized by those skilled in the art to enhance expression efficiency in plants. For example, polynucleotides may be synthesized using optimization of preferred codons of the target plant to enhance expression efficiency in the target plant.

The invention further provides a protein coded by the ZmKOB1 gene, and the amino acid sequence of the protein is shown in (a) or (b):

(a) And an amino acid sequence shown as SEQ ID No. 2; or

(b) And protein variants derived from the amino acid sequence shown in SEQ ID No.2 by substitution, deletion or/and insertion of one or more amino acid residues and still having the function or activity of a glycosyltransferase.

The protein variants of the invention may be produced by genetic polymorphism or by human manipulation, such manipulations being generally known in the art. For example, the amino acid sequence variants or fragments may be prepared by mutation of the DNA, wherein methods for mutagenesis or altering of polynucleotides are well known in the art. Among these, conservative substitutions are substitutions of one amino acid residue for another with similar properties.

By "variant" is meant a substantially similar sequence, and for polynucleotides, a variant comprises a deletion, insertion, or/and substitution of one or more nucleotides at one or more sites in the native polynucleotide. For polynucleotides, conservative variants include those that do not alter the encoded amino acid sequence due to the degeneracy of the genetic code. Naturally occurring variants such as these can be identified by existing molecular biology techniques. Variant polynucleotides also include polynucleotides of synthetic origin, for example, variants of polynucleotides which still encode the amino acid sequence shown in SEQ ID No.2 by site-directed mutagenesis or by recombinant means (e.g., DNA shuffling). One skilled in the art can screen or evaluate the function or activity of a protein encoded by a variant polynucleotide by the following molecular biotechnology means: DNA binding activity, interactions between proteins, activation of gene expression in transient studies or effects of expression in transgenic plants, etc.

The invention also provides a recombinant plant expression vector containing the ZmKOB1 gene and a host cell containing the recombinant plant expression vector.

The invention further constructs a proZmUbi which is driven by the Ubi promoter, wherein ZmKOB1-GFP vector; transforming a maize inbred line C01 by an agrobacterium-mediated method; the transgenic line shows the phenotype of increased row grain number, ear length and ear grain weight, and shows that ZmKOB1 plays an important role in regulating and controlling the agronomic characters of the corn, such as row grain number, ear length, ear grain weight and the like.

In order to further illustrate a molecular mechanism of the ZmKOB1 gene for regulating the development of the female ear, the ZmKOB1 gene in the corn is knocked out by further utilizing a CRISPR/Cas9 transgenic technology, and a ZmKOb1 mutant is obtained through genotype identification, wherein the mutant plant shows phenotypes such as reduced row grain number, shortened ear length, reduced ear grain weight and the like; therefore, the ZmKOB1 gene is determined to have the function of regulating the maize ear trait.

The nucleotide sequence of the ZmKOB1 gene is shown in SEQ ID No.1, and the amino acid sequence of the encoded protein is shown in SEQ ID No. 2.

The invention discloses a recombinant expression vector containing the ZmKOB1 gene and a recombinant host cell containing the recombinant expression vector.

In conclusion, the key gene ZmKOB1 for regulating and controlling the female ear fruit setting traits of the corn is identified, wherein the regulation and control of the female ear fruit setting traits of the corn comprises the increase of the row grain number, the ear length or the ear grain weight of the corn, and the key gene ZmKOB1 is applied to the genetic improvement of the row grain number, the ear length and the ear grain weight of hybrid seeds so as to improve the grain yield of the hybrid seeds; therefore, the ZmKOB1 gene can improve the high maturing rate of the maize female ear and further culture a new high-yield maize variety.

Thus, the invention provides a method for increasing the weight of the row grain number, ear length or ear weight of corn or promoting the development of the female ear of corn: constructing a plant recombinant expression vector containing a ZmKOB1 gene; transforming the plant recombinant expression vector into corn, and performing overexpression on the ZmKOB1 gene in the corn.

Correspondingly, the invention also provides a method for reducing the weight of the row grain number, the ear length or the ear grain weight of the corn or delaying or blocking the development of the corn ear, which comprises the following steps: constructing a knockout vector or a gene editing vector of the ZmKOB1 gene; and (3) transforming the constructed gene knockout vector or gene editing vector into a corn plant, and knocking out or mutating the ZmKOB1 gene in the corn.

Wherein the gene editing vector of the ZmKOB1 gene or the gene knockout vector of the ZmKOB1 gene can be obtained according to a conventional construction method in the art.

As a preferred embodiment, the present invention provides a method for constructing a gene editing vector for the ZmKOB1 gene, comprising:

(1) Preparation of sgRNA expression cassette

Fusing the target sequences of ZmKOB1 genes shown in SEQ ID No.5 and SEQ ID No.6 with the sgRNA framework sequence to obtain two sgRNA expression cassettes;

(2) Cloning the hSpCas9 sequence in human into a pCPB vector to construct a pCPB-ZmUbi vector, wherein the hSpCas9 sequence is a human hSpCas9 sequence;

(3) Inserting two sgRNA expression cassettes into pCPB-ZmUbi, wherein HindIII enzyme cutting sites of hSpCas9 construct a CRISPR/Cas9 gene editing vector.

In addition, the invention also provides a method for improving the high maturing rate of the female ears of the corn or cultivating a high-yield variety of the corn, which comprises the following steps:

constructing a plant recombinant expression vector containing a ZmKOB1 gene; transforming the plant recombinant expression vector into corn, and performing over-expression on the ZmKOB1 gene in the corn; hybridizing ZmKOB1 overexpression strains which show that the number of lines, the length of ears and the weight of the ears are increased with different corn materials, backcrossing and transforming, and improving hybrid seeds to obtain a new high-yield corn variety;

or excavating material containing ZmKOB1 excellent allelic gene type, hybridizing with different corn materials, combining with molecular breeding selection and backcross transformation, and further improving or obtaining a new corn high-yield variety.

For reference, the present invention provides a method comprising a plant recombinant expression vector containing a ZmKOB1 gene, comprising operably linking the ZmKOB1 gene to an expression regulatory element to obtain a plant recombinant expression vector; the plant recombinant plant expression vector can consist of a 5 'end non-coding region, a ZmKOB1 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 protocols described herein, and the protocols for introducing the polynucleotides or polypeptides into plants, 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. The transformed cells can be regenerated into stably transformed plants using conventional methods (McCormick et al plant Cell reports, 1986.5.

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

The key gene ZmKOB1 for regulating and controlling the row grain number, the ear length and the ear grain weight of the corn can increase the row grain number, the ear length and the ear grain weight of the corn, can be used for genetic improvement of the row grain number, the ear length and the ear grain weight of hybrid seeds, and further improves the grain yield of the hybrid seeds; the invention provides a theoretical basis for genetic improvement of the number of corn seeds in a row and the weight of the corn ear, has great breeding application value, and can improve the high seed setting rate of the corn ear so as to cultivate high-yield corn varieties.

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.

The "stringent hybridization conditions" as used herein means conditions of low ionic strength and high temperature known in the art. In general, probes hybridize to their target sequences to a greater extent than to other sequences under stringent conditions (e.g., at least 2-fold over background. Stringent Hybridization conditions are sequence-dependent and will differ under different environmental conditions, longer sequences specifically hybridize at higher temperatures. Target sequences that are 100% complementary to Probes can be identified by controlling the stringency of Hybridization or wash conditions _m ) About 5-10 ℃. T is _m Is the temperature (at a defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence in equilibrium (at T, because the target sequence is present in excess, it is at _m At equilibrium 50% of the probes are occupied). Stringent conditions may be as follows: wherein the salt concentration is less than about 1.0M sodium ion concentration, typically about 0.01 to 1.0M sodium ion concentration (or other salt) at pH 7.0 to 8.3, and the temperature is at least about 30 ℃ for short probes (including but not limited to 10 to 50 nucleotides) and at least about 60 ℃ for long probes (including but not limited to greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, the positive signal can be at least twice background hybridization, optionally 10 times background hybridization. Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5 XSSC and 1% SDS, incubated at 42 ℃; or 5 XSSC, 1% SDS, cultured at 65 ℃, washed in 0.2 XSSC and washed at 65 DEG CWashing in 0.1% SDS. The washing may be for 5, 15, 30, 60, 120 minutes or more.

The "plurality" as referred to in the present invention generally means 2 to 8, preferably 2 to 4, depending on the position of an amino acid residue in the three-dimensional structure of the transcription factor or the kind of an amino acid; the "substitution" refers to the substitution of one or more amino acid residues with different amino acid residues, respectively; the term "deletion" refers to a reduction in the number of amino acid residues, i.e., the absence of one or more amino acid residues, respectively; by "insertion" is meant a change in the sequence of amino acid residues that results in the addition of one or more amino acid residues relative to the native molecule.

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 is 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 or eukaryotic cell, and the host cell may also be a monocotyledonous or dicotyledonous plant cell.

The term "operably connected" refers to a functional connection between two or more elements, which may be contiguous or non-contiguous.

The term "plant recombinant expression vector" means one or more DNA vectors for effecting 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.

Detailed description of the overall solution of the invention

The invention utilizes a proZmUbi driven by a Ubi promoter, zmKOB1-GFP vector and transforms a maize inbred line C01 by an agrobacterium-mediated method. The obtained overexpression transgenic strain expresses the phenotype of grain number, grain length and grain weight increase.

The invention utilizes CRISPR/Cas9 transgenic technology to carry out gene editing on ZmKOB1 gene design targets in corn, so that the ZmKOB1 gene has large fragment (600 bases) deletion and 7 base deletion. Phenotypic investigations show that the zmkob1 mutant plants exhibit phenotypes such as reduced kernel count, shortened ear length, and reduced ear weight.

The invention utilizes ZmKOB1 overexpression lines to hybridize with maize inbred lines, F thereof ₁ The hybrid seeds have longer panicle length, more grain number in rows and increased grain weight in panicle, which indicates that the ZmKOB1 can be used for genetic improvement of the grain number in rows, the grain length and the grain weight in panicle of the hybrid seeds, and further improve the grain yield of the hybrid seeds.

Therefore, the invention provides a theoretical basis for the genetic improvement of the number of the corn seeds, and can improve the high fructification rate of the corn ears so as to cultivate the high-yield corn variety.

Drawings

FIG. 1 is a graph showing the effect of ZmKOB1 gene overexpression on maize row number, ear length and ear weight; (A) The ZmKOB1 overexpression material (OE 3 and OE 4) had longer ear lengths and increased line counts relative to the control. And (B) detecting the expression quantity of the ZmKOB1 overexpression strain. (C) Spike weight was increased for both lines of ZmKOB1 compared to the control.

FIG. 2 is a graph showing the effect of ZmKOB1 gene mutation on row grain number, ear length and ear weight of maize; (A) The zmkob1 mutants (KO #1 and KO # 2) had shorter ear lengths and fewer lines relative to the control. (B) The zmkob1 mutant has a large fragment (600 bases) deletion and 7 base deletion pattern. (C) Spike weight was reduced for the zmkob1 mutant compared to the control.

FIG. 3 is the hybridization and phenotypic analysis of the obtained ZmKOB1 overexpression lines with maize backbone parents (Zheng 58); (A) And relative to negative plant F ₁ (ZHEN 58/CK 4) Positive plants F obtained by crossing ZHEN 58 with OE4 ₁ The number of grains in the row and the ear length increase. (B) And relative to negative plant F ₁ (ZHEN 58/CK 4) at F ₁ The panicle weight of (Zheng 58/OE 4) plants was significantly increased.

Detailed Description

The present invention is further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become more apparent as the description proceeds. 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 ZmKOB1-OE mutant and phenotypic observation test

The test constructs a proZmUbi driven by a Ubi promoter, namely a ZmKOB1-GFP vector and transforms a maize inbred line C01 immature embryo callus by an agrobacterium-mediated method.

The specific construction method comprises the following steps:

the CDS sequence of the amplified ZmKOB1 is connected into the CPB carrier framework by using the CPB carrier as the framework and using the method of infusion.

The primer sequences used were as follows:

OE-ZmKOB1-proUbi-F：

ACTTCTGCAGGGATCCATGGCGGGCTACCGCGGGGG(SEQ ID

No.3)；

OE-ZmKOB1-proUbi-R：

TGCCACCACCGGATCCTGAAAAGGCAGTGACACTTTCAGTCAG

(SEQ ID No.4)；

the transformation method comprises the following steps: maize embryos were transformed using EHA105 Agrobacterium-mediated transformation.

Transgenic plants were obtained via basta resistance screen.

It was observed that the overexpression transgenic lines exhibited a phenotype of increased kernel number, ear length and ear weight (FIG. 1).

Test example 2 construction of maize ZmKOB1 Gene mutant and phenotypic analysis

Construction of CRISPR/Cas9 knockout vector of ZmKOB1 gene

Two targets of ZmKOB1 gene design in corn are subjected to gene editing by using CRISPR/Cas9 transgenic technology.

The specific target sequence of the ZmKOB1 gene (design sgRNA) is designed and screened by utilizing SnapGene Viewer software and homologous sequence alignment, and in order to ensure the gene editing efficiency, each gene selects two optimal target sequences:

the CRISPR/Cas9 target sequence of ZmKOB1 (GRMZM 2G123540, zm00001d 039284) is:

GTAATGGGAAATCAGCTGCA(SEQ ID No.5)；

GGCTATCATTCGTGGCCTCA(SEQ ID No.6)；

these target sequences were introduced into the sgRNA expression cassette, and the hSpCas9 sequence In humans was used with commercial In-

The PCR Cloning Kit is cloned into a pCPB vector to construct a pCPB-ZmUbi vector, wherein the hSpCas9 vector is a promoter. Then, two sgRNA expression cassettes were passed through In-

The HD Cloning Kit is inserted into pCPB-ZmUbi, wherein HindIII enzyme cutting sites of hSpCas9 are inserted into the HindIII enzyme cutting sites, and the finally constructed CRISPR/Cas9 gene editing vector is used for subsequent genetic transformation after being verified to be correct by PCR sequencing.

2. Corn inbred line C01 transformed by constructed CRISPR/Cas9 vector through agrobacterium-mediated method and phenotype analysis of transgenic plant

And transforming the constructed CRISPR/Cas9 gene editing vector of the ZmKOB1 into a maize inbred line C01 by an agrobacterium-mediated method. A mutant with deletion of a large fragment (600 bases) and deletion of 7 bases of the ZmKOB1 gene is obtained. Phenotypic investigations showed that zmkob1 mutant plants exhibited phenotypes such as reduced kernel number, shortened ear length, and reduced ear weight (fig. 2).

Test example 3 hybridization of ZmKOB1 overexpression lines with maize backbone parents and phenotypic analysis

The ZmKOB1 overexpression lines obtained in example 1 were combined with maize backbone parents (ZHEN 58) to form F ₁ And performing phenotype observation at Hainan planting base.

The phenotypic observations are shown in FIG. 3, and relative to negative plant F ₁ (ZHEN 58/CK 4) Positive plants F obtained by crossing ZHEN 58 with OE4 ₁ Significantly increased row grain number, ear length and ear grain weight (FIG. 3); the result shows that the glycosyltransferase ZmKOB1 plays a crucial role in the aspects of regulating and controlling heterosis utilization of corn and the like.

SEQUENCE LISTING

<110> southern China university of agriculture

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Leu Thr Leu Leu Pro Leu Ala Leu Ala Ala Phe Ala Phe Ala Leu Gln

35 40 45

Trp Arg Gly Gly Met Arg Asp Pro Ala Gly Ala Ala Trp Pro Ala Asp

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Ala Gln Arg Phe Pro Gly Met Glu Asn Ser Pro Leu Ser Ser Ser His

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Gly Gly Ala Gly Ser Tyr Phe Ala Val Ser Ser Ser Ser Ser Ser Pro

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Gly Ile Ser Leu Tyr Arg Gly Trp Ser Phe Asp Ser Glu Ser Ala Ile

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Thr Pro Lys Ile Cys Ile Thr Gly Ser Thr Ser Ala Ser Leu His Gln

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Gly Phe Phe Tyr Lys Pro Cys Asn Tyr Glu Leu Phe Val Lys Gln Ser

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Leu Asn Met Glu Met Ala Ile Ile Met Ala Arg Asp Ala Gly Met Asp

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Trp Ile Ile His Leu Asp Thr Asp Glu Leu Ile His Pro Ala Gly Ala

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Arg Glu Tyr Ser Leu Arg Arg Leu Leu Leu Asp Val Pro Asp Asn Val

260 265 270

Asp Met Val Ile Phe Pro Asn Tyr Glu Ser Ser Ile Glu Arg Asp Asp

275 280 285

Ile Lys Asp Pro Phe Thr Glu Val Ser Met Phe Lys Lys Asn Tyr Asp

290 295 300

His Leu Pro Lys Asp Thr Tyr Phe Gly Leu Tyr Lys Glu Ala Thr Arg

305 310 315 320

Gly Asn Pro Asn Tyr Phe Leu Thr Tyr Gly Asn Gly Lys Ser Ala Ala

325 330 335

Arg Val Gln Glu His Leu Arg Pro Asn Gly Ala His Arg Trp His Asn

340 345 350

Tyr Met Lys Ser Pro Asn Glu Ile Lys Leu Glu Glu Ala Ala Ile Leu

355 360 365

His Tyr Thr Tyr Thr Lys Phe Ser Asp Leu Thr Ser Arg Arg Asp Arg

370 375 380

Cys Gly Cys Lys Pro Thr Lys Glu Asp Val Lys Arg Cys Phe Ile Leu

385 390 395 400

Glu Phe Asp Arg Leu Ala Phe Ile Ile Ala Ser Thr Ala Thr Glu Glu

405 410 415

Glu Met Arg Asn Trp Tyr Lys Glu His Val Val Trp Thr Asp Arg Asp

420 425 430

Thr Asn Leu Lys Leu Leu Arg Lys Ser Val Leu Thr Arg Ile Tyr Ala

435 440 445

Pro Met Ala Ile Ile Arg Gly Leu Lys Glu Ser Gly Val Phe Thr Asn

450 455 460

Ala Val Thr Ser Ala Lys Ala Gln Ser Lys Thr Lys Ser Ser Asn Met

465 470 475 480

Gly Leu Gly Asn Lys Asp Pro Ile Gln Pro Asn Gly Thr Ala Gly Gln

485 490 495

Ser Thr Leu Glu Gly Ser His Gly Lys Leu Gln Ala Thr Val Arg Lys

500 505 510

Ile Leu Glu Met Val Asp Thr Gln Glu Glu Ala Ala Met Pro Pro Met

515 520 525

Ser Pro Pro Gly Phe Leu Glu Leu Thr Glu Ser Val Thr Ala Phe Ser

530 535 540

<210> 3

<211> 36

<212> DNA

<213> Artifical sequence

<400> 3

acttctgcag ggatccatgg cgggctaccg cggggg 36

<210> 4

<211> 43

<212> DNA

<213> Artifical sequence

<400> 4

tgccaccacc ggatcctgaa aaggcagtga cactttcagt cag 43

<210> 5

<211> 20

<212> DNA

<213> Artifical sequence

<400> 5

gtaatgggaa atcagctgca 20

<210> 6

<211> 20

<212> DNA

<213> Artifical sequence

<400> 6

ggctatcatt cgtggcctca 20

Claims

1. A ZmKOB1 gene isolated from maize, wherein the polynucleotide sequence of the CDS is (a), (b), (c), (d) or (e):

(a) A polynucleotide shown as SEQ ID No. 1; or

(b) A polynucleotide encoding the amino acid shown in SEQ ID No. 2; or

(c) A polynucleotide capable of hybridizing to the complement of the polynucleotide of SEQ ID No.1 under stringent hybridization conditions; or

(d) A polynucleotide having at least 90% or more homology to the polynucleotide shown in SEQ ID No. 1; or

(e) A polynucleotide variant having deletion, substitution or insertion of one or more bases based on the polynucleotide shown in SEQ ID NO.1, wherein the protein encoded by the polynucleotide variant still has the function or activity of glycosyltransferase.

2. The protein encoded by ZmKOB1 gene according to claim 1, wherein the amino acid sequence of the protein is represented by (a) or (b):

(a) An amino acid sequence shown as SEQ ID No. 2; or

(b) A protein variant derived from the amino acid sequence shown in SEQ ID No.2 by substitution, deletion or/and insertion of one or more amino acid residues which still has the function or activity of a glycosyltransferase.

3. An expression vector comprising the ZmKOB1 gene according to claim.

4. The ZmKOB1 gene of claim 1 for use in modulating maize ear set trait or ear development.

5. The use according to claim 4, wherein the modulation of maize female ear seed set traits comprises increasing maize row number, ear length or ear weight; or the regulation and control of the maize female ear seed setting traits comprise reduction of the row grain number, the ear length or the weight of the ear grain weight of the maize.

6. The use according to claim 4, wherein said modulating development of a maize ear is promoting development of a maize ear; or the regulation and control of the development of the maize female ear is to delay or block the development of the maize female ear.

7. A method for increasing the weight of row size, ear length or ear weight of corn or promoting development of female ears of corn, comprising: constructing a plant recombinant expression vector containing a ZmKOB1 gene; transforming the plant recombinant expression vector into corn to ensure that the ZmKOB1 gene is over-expressed in the corn.

8. A method for reducing the weight of row grain number, ear length or ear weight of corn to retard or arrest the development of the ear of corn, comprising: constructing a knockout vector or a gene editing vector of the ZmKOB1 gene; and transforming the constructed gene knockout vector or gene editing vector into a corn plant, and knocking out or mutating the ZmKOB1 gene in the corn.

9. The method according to claim 8, wherein the construction method of the ZmKOB1 gene editing vector comprises:

(1) Preparation of sgRNA expression cassette

Respectively fusing target sequences of ZmKOB1 genes shown in SEQ ID No.5 and SEQ ID No.6 with sgRNA framework sequences to obtain two sgRNA expression cassettes;

(2) Cloning the hSpCas9 sequence in human into a pCPB vector to construct a pCPB-ZmUbi vector, wherein the hSpCas9 vector is a promoter of the hSpCas;

(3) And (3) inserting the two sgRNA expression cassettes into pCPB-ZmUbi, wherein the HindIII enzyme cutting sites of hSpCas9 construct a CRISPR/Cas9 gene editing vector.

10. A method for improving the high seed setting rate of corn ears or cultivating high-yield corn varieties is characterized by comprising the following steps: constructing a plant recombinant expression vector containing a ZmKOB1 gene; transforming the plant recombinant expression vector into corn, and performing overexpression on the ZmKOB1 gene in the corn; hybridizing ZmKOB1 overexpression strains with different corn materials and backcrossing and transforming the strains to improve hybrid seeds so as to obtain new high-yield corn varieties;

or excavating a material containing ZmKOB1 excellent allelic gene types, hybridizing with different corn materials, combining with molecular breeding selection and backcross transformation to obtain a new corn high-yield variety.