CN112175969A

CN112175969A - Preparation and application of corn ZmFKF1 gene and gene editing mutant thereof

Info

Publication number: CN112175969A
Application number: CN202011087619.2A
Authority: CN
Inventors: 杨敏; 王翠玲; 胥华伟; 杨护; 魏岳荣
Original assignee: Pomology Research Institute Guangdong Academy of Agricultural Sciences
Current assignee: Pomology Research Institute Guangdong Academy of Agricultural Sciences
Priority date: 2020-10-13
Filing date: 2020-10-13
Publication date: 2021-01-05
Anticipated expiration: 2040-10-13
Also published as: CN112175969B

Abstract

The invention belongs to the technical field of biological engineering, and particularly relates to a corn ZmFKF1 gene, and further discloses preparation and application of a gene editing mutant thereof. The ZmFKF1 gene disclosed by the invention takes corn 'B104' as a material to obtain a CDS sequence of the ZmFKF1 gene, utilizes a CRISPR/Cas9 gene editing technology, takes corn 'B104' immature embryos as a receptor material, and utilizes an agrobacterium-mediated method to successfully carry out site-directed editing on the corn ZmFKF1 gene to obtain 3 homozygous lines mutated at a target site, and the flowering phenotype comparison with wild-type 'B104' shows that the time for carrying out site-directed editing on the ZmFKF1 mutant for drawing male, spinning and powder scattering is delayed, and the expression of key flowering genes in the lines is also reduced, so that the ZmFKF1 gene plays an important role in a corn flowering pathway, the regulation and control function of the ZmFKF1 gene in the corn flowering pathway is reported for the first time, and the stage is provided for corn molecule breeding and genetic improvement.

Description

Preparation and application of corn ZmFKF1 gene and gene editing mutant thereof

Technical Field

The invention belongs to the technical field of biological engineering, and particularly relates to a corn ZmFKF1 gene, and further discloses preparation and application of a gene editing mutant thereof.

Background

Corn is used as an important grain and feed crop in China, and plays an important role in national economy and daily life of people. However, due to the problem of limited breeding germplasm, great challenges are still faced in the aspects of excellent variety breeding and yield improvement. Flowering is a key stage of a plant growth process, and is also an urgent problem to be solved in the practical process of introducing tropical corns into temperate zones for planting. Therefore, the research on key genes participating in the corn flowering approach can further regulate and control the flowering process of corn plants, and the method has important significance on corn molecular breeding and genetic improvement.

FLAVINBINDING KELCH REPEAT F-BOX1(FKF1) belongs to the family of the ZTLS blue light receptor, and is a protein comprising three functional domains of LOV domain, F-BOX motif and Kelch repeat structure. FKF1 has been shown to play an important role in the flowering process in a variety of plants. In the model plant Arabidopsis, the action mechanism of FKF1 is studied more deeply, and the FKF1-GI-CDF1- - -CO-FT plays a role in photoperiod flowering, namely: the LOV domain of FKF1 interacts with GI to form a complex, the stability of which is regulated by blue light; CDF1 (cycle DOF FACTOR 1) is a transcription repressor of CO, and is directly combined with a promoter of CO to block the expression of CO and inhibit flowering; both FKF1 and GI can interact with CDF1, which degrades CDF1 under the action of the ubiquitin-proteasome system, thereby promoting CO expression. Under the condition of long day, because the expression of FKF1 and GI is synchronous, enough FKF1-GI complex can be formed, the transcription of CO can be activated in the day, and the stability of CO protein depends on light, the accumulation of CO protein can reach a peak value in the evening under the existence of light, and the peak value acts on downstream FLOWERING gene FLOWERING LOCUST (FT), thereby promoting the FLOWERING of plants. In addition, FKF1 protein also stabilizes CO protein by interacting with CO protein via the LOV domain independent of interaction with GI protein. Therefore, in arabidopsis thaliana, the FKF1 protein can regulate the expression of CO through various ways after transcription and translation, further promote the expression of FT, and promote the flowering of plants. Under short day conditions, FKF1 and GI expression are not synchronized and less FKF1-GI complex is formed until midnight CO transcript accumulation reaches a higher level, but because there is no light, CO protein is still at a lower level, resulting in a lower level of downstream FT expression and failure to promote flowering. Similarly, OsFKF1 was also found and studied intensively in rice, which is a short-day plant, and it was confirmed that OsFKF1 up-regulates Ehd1 by down-regulating the expression of both genes Ghd7 and Ehd2, and that Ehd1 continues to promote rice flowering by up-regulating Hd3a/RFT1 genes. However, flowering genes cloned in maize are relatively rare compared to Arabidopsis and rice, and most candidate genes are obtained from homology analysis. Major QTLs associated with flowering-time or photoperiod exist in region 10.04 on chromosome 10, 9.05 on chromosome 9 and 4.09 on chromosome 4 of maize. Currently, ZmCCT in the 10 th chromosome 10.04 region can greatly shorten the flowering time and effectively weaken the photoperiod sensitivity of corn. ZmPRR73, a candidate gene participating in flowering in the 9.05 region of chromosome 9, is preliminarily determined by Zhaoshi et al. However, no studies have been reported on the 4.09 region of chromosome 4, in which ZmFKF1 is an important candidate gene in this region that is likely to be involved in maize flowering. Previously, Liuling et al carried out molecular evolution and correlation analysis studies of the blue light response rhythm gene ZmFKF1 in the maize photoperiod pathway, but did not carry out extensive studies on the specific role of ZmFKF 1.

The newly developed gene editing technology CRISPR/Cas9 system provides a more convenient and effective way for the research of gene functions. The CRISPR/Cas9 is a novel technology for efficiently editing genes at fixed points after TALLEN and ZFN, and has become a technical hotspot for extensive researchers to try to deeply research gene functions in recent years. At present, the CRISPR/Cas9 system successfully realizes gene editing on Arabidopsis, rice, wheat, cotton, tomato, poplar, apple, watermelon, grape, cabbage, orange and other plants. In corn, the group of subjects uses Zmzb7 with known functions to show that the CRISPR/Cas9 system can be used for the gene function research of corn, but the research of unknown gene functions by using the technology is not reported.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to provide a corn ZmFKF1 gene, and the CRISPR/Cas9 gene editing technology is used for carrying out site-directed editing on the ZmFKF1 gene, so that the regulation and control effect of ZmFKF1 on the corn flowering process is researched;

in order to solve the technical problems, the invention provides a corn ZmFKF1 gene, wherein the ZmFKF1 gene comprises a nucleotide sequence shown as SEQ ID No: 1.

Preferably, the nucleotide sequence of the CDS region of the ZmFKF1 gene is as shown in SEQ ID No: 1 is shown.

The invention also discloses a mutant edited by the maize ZmFKF1 gene by utilizing CRISPR/Cas9 technology.

The invention also discloses a gene editing expression vector containing the corn ZmFKF1, and the expression vector is pYLCISPR/Cas 9-ZmFKF 1-T1.

The invention also discloses a method for constructing the gene editing expression vector, which comprises the steps of selecting a nucleotide sequence on the first exon of the ZmFKF1 gene as a target site, connecting the nucleotide sequence to a pYLgRNA-U6b vector, and further connecting the nucleotide sequence to a pYLCRISPR/Cas9 binary vector.

Specifically, in the construction method of the expression vector, the target site selects 20 nucleotide sequences on the first exon of the ZmFKF1 gene, and the sequence structure is as follows: 5'-AGGTGGACGCGGAGCCGGGA-3' are provided.

The invention also discloses a recombinant bacterium containing the ZmFKF1 editing expression vector.

The invention also discloses a method for constructing the recombinant strain, which comprises the step of introducing the ZmFKF1 edited expression vector into an agrobacterium host strain so as to ensure that the vector is effectively expressed in the host.

Specifically, the method further comprises the steps of transferring the vector into escherichia coli DH5 alpha competent cells, picking a single colony to perform colony PCR positive detection (primer SP-L1/SP-R), performing sequencing verification on PCR positive bacteria liquid, and introducing a plasmid with correct sequencing into agrobacterium-infected competent cells EHA 105.

Specifically, the step of obtaining the ZmFKF1 gene editing positive plants provides a screening process for obtaining resistant callus through two screening processes, firstly, a first round of resistance screening is carried out for 14 days by using a gene containing cefuromycin (250mg/L) and PPT (5mg/L), secondly, the concentration of cephalosporin is not changed, the concentration of PPT is increased to 10mg/L, a second round of resistance screening is carried out for 14 days, the resistant callus is obtained, then, germination is induced, when 2-3 leaves grow on the plantlets and main stems exist, the plantlets are transferred to a rooting culture medium for rooting, resistant regenerated plantlets are obtained, and 0.125% (V/V) glufosinate ammonium is used for smearing screening at the 6-7 leaf stage, sensitive plants are removed, and good resistant regenerated plants are obtained.

The invention also discloses application of the corn ZmFKF1 gene in the field of corn breeding improvement and cultivation.

The invention also discloses application of the mutant in the fields of corn breeding improvement and cultivation.

After the maize ZmFKF1 gene is edited in a fixed point, the mutant shows an obvious late-flowering phenotype, the ZmFKF1 gene positively regulates and controls a maize flowering way, and the role of the ZmFKF1 gene in the process of regulating and controlling maize flowering, which is an important agronomic trait, is reflected.

The corn ZmFKF1 gene is prepared by taking corn 'B104' as a material to obtain a corn ZmFKF1 gene CDS sequence, using a CRISPR/Cas9 gene editing technology, using corn 'B104' immature embryos as a receptor material and using an agrobacterium-mediated method to successfully carry out site-directed editing on the corn ZmFKF1 gene to obtain 3 homozygous strains mutated at target sites, and comparing flowering phenotypes with wild type 'B104', the time for spaying, spitting and scattering of the ZmFKF1 site-directed editing mutant is found to be delayed, and the expression of key flowering genes in the strains is also reduced, so that the ZmFKF1 gene plays an important role in a corn flowering pathway to define the biological function of ZmFKF1, the regulation and control function of the ZmFKF1 gene in the corn flowering pathway is reported for the first time, and the basis is provided for corn molecular breeding and genetic improvement.

According to the scheme, corn 'B104' is used as a material, the ZmFKF1 gene is used as a target point, the fixed point editing is carried out by using a CRISPR/Cas9 technology, the ZmFKF1 gene can be directionally and efficiently modified from a molecular genetics level, the ZmFKF1 gene editing mutant is obtained, the function of the ZmFKF1 in a corn flowering way is researched through phenotypic analysis, gene expression detection and the like, the function of the unknown corn gene can be revealed by using the technology, a new gene resource is provided for researching and enriching the corn flowering way, and the method has important significance for the field of molecular breeding.

Drawings

In order that the present disclosure may be more readily and clearly understood, the following detailed description of the present disclosure is provided in connection with specific embodiments thereof and the accompanying drawings, in which,

FIG. 1 is a schematic diagram of the gRNA target site and the T-DNA region of pYLCRISPR/Cas9-ZmFKF1-T1 plasmid; wherein, A: the position of the target site in ZmFKF 1; b: pYLCRISPR/Cas9-ZmFKF1-T1 plasmid T-DNA region schematic;

FIG. 2 is a diagram of the transformation process of maize ` B104 ` immature embryos; wherein, A-immature embryo, B-primary callus, C-resistant callus, D-regenerated resistant bud, E-regenerated resistant seedling;

FIG. 3 is a drawing showing the identification and sequencing analysis of ZmFKF1 gene-edited plants; wherein, the positive identification of the A-ZmFKF1 gene editing plant and the homozygote sequencing result of the B-ZmFKF1 gene editing;

FIG. 4 shows the prediction of the ZmFKF1 domain and the analysis of the amino acid sequence encoding ZmFKF1 in the mutant; wherein, A-ZmFKF1 protein domain prediction result; B-ZmFKF1 editing mutant amino acid sequence prediction analysis and alignment result;

FIG. 5 is a diagram showing the phenotypic analysis of ZmFKF1 gene-edited plants and the expression of key genes; among them, the phenotype of A-C7-1 and 'B104' at 67 days after sowing, the phenotype of B-C7-1 and 'B104' at 70 days after sowing, the expression of flowering key genes in C-different gene-type materials.

Detailed Description

The invention is given by the following detailed examples. From the following description and these examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

In the following examples of the present invention, the experimental materials involved were subjected to conventional planting and field management.

The sgRNA intermediate vector pYLgRNA-U6b and the CRISPR/Cas9 binary vector were offered by Sizuki, the university of south China agricultural, Liquidambar, and primers and sequencing used in the experiments were completed by Biotechnology (Shanghai) GmbH.

Bsa I was purchased from NEB, T4 DNA ligase and PrimeSTAR GXL DNA Polymerase from BaoBioengineering (Dalian) Co., Ltd., KOD FX was purchased from Toyobo Biotechnology Co., Ltd., and the rest of the molecular reagents were purchased mainly from Tiangen Biochemical technology (Beijing) Co., Ltd., and Biochemical engineering (Shanghai) Co., Ltd.

Escherichia coli (Escherichia coli) competent cell DH5 α was purchased from Tiangen Biochemical technology (Beijing) Ltd; agrobacterium-competent cells EHA105 were purchased from Beijing Huayue ocean biology, Inc.

In the following examples of the present invention, the cloning, vector construction and identification primers involved are shown in Table 1 below.

TABLE 1 cloning, vector construction and identification primers used in the experiments

Example 1 cloning of CDS region of ZmFKF1 Gene, target selection and editing vector construction

The cloning and expression method of ZmFKF1 gene in this example includes the following steps:

(1) cloning of CDS region of ZmFKF1 Gene

The Primer ZmFKF1-F/ZmFKF1-R (shown in Table 1) was designed using Primer Premier 5.0 with reference to the Maize ` B104 ` ZmFKF1 sequence of the Maize GDB (https:// www.maizegdb.org /) website.

The RNA of maize 'B104' leaf was extracted by Trizol method, and genomic DNA contamination of total RNA was removed by DNaseI (Takara, Japan) according to a conventional method, using PrimeScripffM II 1^stStrand cDNA Synthesis Kit reverse transcription Kit (Takara, Japan) reverse transcribes the extracted total RNA into single-stranded cDNA. Using cDNA as template and ZmFKF1-F/ZmCDF1-R as primer (see Table 1), CDS cloning of coding sequence was performed, PCR product was electrophoresed through 1.5% agarose gel, and the result was detected and recorded by gel imaging system (Bio-Rad). And recovering the PCR product, sequencing the PCR product by an engineering bioengineering (Shanghai) corporation, obtaining a CDS sequence of the ZmFKF1 by sequencing, wherein the sequence size is 1857 bp, and determining that the sequence of the CDS region of the obtained ZmFKF1 gene is shown as SEQ ID No: 1 is shown.

(2) ZmFKF1 gene target selection and editing vector construction

Target site selection was performed in conjunction with the online site CRISPR-GE (http:// skl.scau.edu.cn /) based on the CRISPR/Cas9 system recognizing the characteristic of about 20 nucleotide sequences upstream of the Protospacer Adjacent Motif (PAM).

Comparing and analyzing the designed target sequence in a maize reference genome, excluding non-specific target sites, and finally screening out a target site on the 1 st exon of ZmFKF1, wherein the sequence is as follows: 5'-AGGTGGACGCGGAGCCGGGA-3', the sequence has no potential off-target sites and is named ZmFKF1-T1, and the PAM sequence is 5 '-TGG-3'. Referring to the methods of Changchang et al (Changchang, Maxingliang, Xianjirong, Liotang, Liu Guanguang), the operation method of construction and mutation analysis of the multiple gene editing vector of the plant criprpr/Cas 9, China science, Life science, 2018,48(7): 783-:

(1) constructing pYLgRNA-U6b vector containing ZmFKF1-T1 target spot: the adaptor primer (FKF-U6b-F/FKF-U6b-R) (Table 1) was denatured and cooled to room temperature to complete annealing, and then ligated to the pYLgRNA-U6b vector by one step of enzymatic ligation, and first round PCR amplification comprising 2 reactions was performed using this as a template: reaction 1 uses U-F/FKF-U6b-R (shown in table 1) as a primer, and reaction 2 uses FKF-U6b-F/gR (shown in table 1) as a primer for amplification; continuously taking the two PCR amplification products as templates, and amplifying by using a specific primer Pps-R/Pgs-L (shown in table 1) to obtain an sgRNA expression cassette ZmFKF1-T1-sgRNA containing ZmFKF1-T1 targets;

(2) cloning the ZmFKF1-T1-sgRNA expression cassette onto pYLCRISPR/Cas9 vector: mixing a ZmFKF1-T1-sgRNA expression cassette obtained by PCR amplification with pYLCRISPR/Cas9 plasmid, and carrying out a side enzyme trimming and connecting reaction in a PCR instrument by using Bsa I enzyme and T4 DNA ligase;

(3) transformation and sequencing verification: transforming the ligation product into DH5 alpha competent cells, picking single colonies for colony PCR positive detection (primer SP-L1/SP-R, table 1), and carrying out sequencing verification on PCR positive bacteria liquid to obtain a pYLCISPR/Cas 9-ZmFKF1-T1 expression vector edited by ZmFKF1 gene; and then extracting a plasmid, introducing the plasmid into the agrobacterium-infected competent cell EHA105, and using the obtained agrobacterium for the subsequent transformation of maize immature embryos.

Example 2 genetic transformation of maize ` B104 ` immature embryos and acquisition of resistant plants

As shown in FIG. 2, the genetic transformation of maize ` B104 ` immature embryos and the preparation of resistant plants in this example comprise the following steps:

(1) preparation of Agrobacterium liquid and maize 'B104' immature embryo

And (3) overnight culturing the activated agrobacterium liquid, and adjusting the OD600 of the liquid to be 0.3-0.4. Taking corn ears 9-12 days after pollination, removing outer bracts, sterilizing in 70% ethanol for 1 minute, and then placing on a super clean bench for drying. 2/3 of the endosperm of the corn kernel was cut off with a scalpel, and the young embryo was peeled off (200 young embryos in total, transformed in two times). Immature maize embryos were co-cultured with agrobacterium for 3 days at 22 ℃ in the dark, after which they were placed at 28 ℃ in the dark for two rounds of resistance selection: first, a first round of resistance screening was performed for 14 days in the presence of cefamycin (250mg/L) and PPT (5 mg/L); secondly, the cephalosporin concentration is unchanged, the PPT concentration is increased to 10mg/L, the second round of resistance screening is carried out for 14 days, resistant callus is obtained, then, the bud is induced, and when the plantlet grows 2-3 leaves and has a main stem, the plantlet is transferred to a rooting culture medium to root, and a resistant regeneration plantlet is obtained.

(2) Acquisition of resistant plants

And (3) field planting the obtained resistance regeneration seedlings until 6-7 leaf stages, smearing and screening by using 0.125% (V/V) glufosinate ammonium, observing the growth condition of leaves for about one week, and removing sensitive plants to obtain a regeneration strain with good resistance.

Example 3 molecular detection of transgenic plants

The molecular detection of the transgenic plant in the embodiment comprises the following steps:

(1)T₀identification of transgenic positive plants

Respectively taking 'B104' wild type and resistant regeneration strain leaves as materials, and extracting T by adopting a CTAB method₀DNA of the generation plant, Cas9 specific primer Cas9-F/Cas9-R (shown in table 1) is used for carrying out vector specific fragment amplification, and the plant capable of amplifying target fragment is T₀Transgenic positive plants are generated.

(2)T₂Generation of ZmFKF1 Gene editing mutants

T obtained by screening₀Obtaining T after selfing and fructification of the generation mutant strain₁Generation of seed, mixing T₁After sowing seeds, transplanting seedlings, extracting leaf genome DNA, amplifying a target design primer ZmCri-F/ZmCri-R (shown in table 1), performing PCR amplification, sequencing and comparison analysis by using the primer, determining the mutation type of the ZmFKF1 gene editing mutant, and screening homozygous mutant single plants. Selfing and breeding the screened ZmFKF1 homozygous mutant to obtain T₂The Generation ZmFKF1 Gene editing mutants are tabulatedAnd (4) type analysis.

The following table 2 shows the analysis of mutation types of ZmFKF1 gene editing mutants, wherein the PAM sequence is shown in bold font, the inserted base is shown in underlined font, + the base insertion, -the base deletion is shown, and-the wild type is shown without change.

TABLE 2 analysis of mutation types of ZmFKF1 Gene editing mutants

FIG. 3 is a diagram showing the identification and sequencing analysis of ZmFKF1 gene-edited plants of this example; wherein, the A-ZmFKF1 gene edits positive identification of plants, and the B-ZmFKF1 gene edits homozygote sequencing results.

Example 4 amino acid sequence prediction analysis of ZmFKF1 editing mutants

The amino acid sequence prediction analysis of the ZmFKF1 editing mutant described in this example specifically includes the following steps:

the ZmFKF1 amino acid sequence was downloaded from NCBI and the domain of ZmFKF1 was predicted online on SMART (http:// SMART. ZmFKF1 comprises 1 PAS (LOV) domain encoded by amino acids 37-107; 1F-box domain encoded by amino acids 205-253; 4 Klech repeated domain, respectively by 300-351, 352-405, 415-466, 517-566 amino acid coding; and simultaneously, the amino acid sequences of the C1-6, C2-12, C3-3, C5-7, C6-2 and C7-1 mutant encoding proteins are predicted and compared, and encoding amino acid differences are analyzed.

FIG. 4 shows the prediction of the ZmFKF1 domain and the analysis of the amino acid sequence encoding ZmFKF1 in the mutant; wherein, A-ZmFKF1 protein domain prediction result; B-ZmFKF1 editing mutant amino acid sequence prediction analysis and alignment results.

Example 5 phenotypic analysis of transgenic plants and detection of expression level of flowering-related Gene

The phenotype analysis of the transgenic plant and the detection of the expression level of the flowering related gene described in this embodiment specifically include the following steps:

(1) phenotypic analysis of transgenic plants

And (3) counting and analyzing flowering phase characters: the obtained ZmFKF1 gene editing homozygous mutant and wild type 'B104' field planting were subjected to flowering phenotype statistics and analysis. 30 plants are randomly selected from each transgenic line and wild type 'B104', phenotype analysis and statistics are carried out on the time of tasseling, spinning and pollen scattering, the experiment is repeated three times, and the average value is taken. The tassel drawing time represents the days after sowing until the tip of the tassel is exposed by about 2 cm; the pollen scattering time represents the days from sowing to pollen scattering of the tassels; the silking time indicates the number of days from sowing until the plant filaments protrude from the bracts.

(2) Expression level detection of flowering related key gene in transgenic plant

Detection of corn flowering key genes: the ZmFKF1 gene is used for editing homozygous mutant, cDNA of 'B104' corn material is used as a template, corn Ubi is used as an internal reference gene, real-time quantitative PCR is used for detecting the expression conditions of important genes related to flowering in different gene type materials, including ZmGI, conz1 and ZmCN8 genes which are proved to be related, and primers are shown in Table 1. The experiment was repeated three times. By use of 2^-ΔΔCTThe method calculates the relative expression level of the gene.

FIG. 5 is a diagram showing phenotype analysis of ZmFKF1 gene-edited plants and expression of key genes, wherein A-C7-1 and 'B104' are expressed at 67 days after sowing, B-C7-1 and B104 are expressed at 70 days after sowing, and C is an expression of flowering key genes in different gene-type materials.

Table 3 shows the statistics of the stamen-pulling time, the silking time and the pollen-scattering time of the maize plants with different genotypes, and notes: indicates significant differences at the 0.05 level.

TABLE 3 statistics of maize tasseling time, silking time and pollen scattering time for different genotype plants

Genotype(s)	Time of taking out male	Time of powder scattering	Time of laying
				B104	62.70±1.37	67.87±1.04	70.53±1.25
C2-12	63.43±1.04*	69.13±1.36*	72.57±1.65*
				C6-2	64.07±0.98*	69.37±1.56*	71.90±1.22*
C7-1	63.96±1.30*	69.04±1.81*	72.33±1.12*

Therefore, the CDS sequence of the corn ZmFKF1 gene is obtained, the corn ZmFKF1 gene can be successfully subjected to site-directed editing by using a CRISPR/Cas9 gene editing technology to obtain 3 homozygous lines mutated at a target site, and compared with a wild type 'B104' flowering phenotype, the site-directed editing mutant of ZmFKF1 is found to have delayed stamina drawing, silking and pollen scattering time, and the expression of a key flowering gene in the lines is also reduced, so that the ZmFKF1 plays an important role in a corn flowering pathway.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Claims

1. A maize ZmFKF1 gene, wherein the ZmFKF1 gene comprises the amino acid sequence set forth in SEQ ID No: 1.

2. The maize ZmFKF1 gene of claim 1, wherein the nucleotide sequence of the CDS region of the ZmFKF1 gene is as set forth in SEQ ID No: 1 is shown.

3. A mutant edited by the maize ZmFKF1 gene of claim 1 or 2.

4. A gene-editing expression vector comprising the maize ZmFKF1 of claim 1 or 2, wherein the ZmFKF 1-editing expression vector is pYLCRISPR/Cas9-ZmFKF 1-T1.

5. A method for constructing the gene editing expression vector of claim 4, which comprises the steps of selecting a nucleotide sequence on the first exon of the ZmFKF1 gene as a target site, ligating it to pYLgRNA-U6b vector, and further ligating it to pYLCRISPR/Cas9 binary vector.

6. The method for constructing a gene editing expression vector according to claim 5, wherein the target site selects 20 nucleotide sequences on the first exon of the ZmFKF1 gene, and the sequence structure thereof is as follows: 5'-AGGTGGACGCGGAGCCGGGA-3' are provided.

7. A recombinant bacterium comprising the ZmFKF1 editing expression vector of claim 4.

8. A method for constructing the recombinant strain of claim 7, comprising the step of introducing the ZmFKF1 editing expression vector of claim 4 into an Agrobacterium host strain, so that the vector is efficiently expressed in the host.

9. The use of the maize ZmFKF1 gene of claim 1 or 2 in the field of maize breeding improvement and breeding.

10. The use of the mutant of claim 3 in the field of maize breeding improvement and breeding.