Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (4): 696-707.doi: 10.3864/j.issn.0578-1752.2021.04.003

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Using CRISPR/Cas9-mediated Targeted Mutagenesis of ZmFKF1 Delayed Flowering Time in Maize

YANG Min1(),XU HuaWei2,WANG CuiLing2,YANG Hu1,WEI YueRong1()   

  1. 1Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences/ Key Laboratory of South Subtropical Fruit Biology and Genetic Resource Utilization (MOA)/Guangdong Province Key Laboratory of Tropical and Subtropical Fruit Tree Research, Guangzhou 510640
    2College of Agriculture, Henan University of Science and Technology, Luoyang 471000, Henan
  • Received:2020-07-25 Accepted:2020-09-23 Online:2021-02-16 Published:2021-02-16
  • Contact: YueRong WEI E-mail:minyang_0123@126.com;weid18@163.com

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Abstract:

【Objective】FKF1 is a key gene that plays an important role in many plant flowering pathways. In order to study the function of FKF1 in maize, ZmFKF1 editing mutants were obtained by using CRISPR/cas9 technology. In this study, we used these mutants as materials to clarify the role of ZmFKF1 in maize flowering pathway through phenotypic analysis and the expression analysis of key flowering genes. The result provide a theoretical basis for molecular breeding and genetic improvement of maize.【Method】 ZmFKF1 gene was cloned from B104, and its structure was determined by sequence alignment. The targeted sequences of ZmFKF1 were designed according to the principle of CRISPR/Cas9 technology, then these targeted sequences were compared and analyzed in maize reference genome, and the non-specific target site was excluded. Finally, ZmFKF1-T1 on exon 1 of ZmFKF1 was selected to construct CRISPR/cas9 gene editing expression vector. At the same time, B104 immature embryos were selected as explants to transform by Agrobacterium-mediated genetic transformation, the resistant calli were obtained through resistance screening, and then buds and roots were induced. The T0 generation ZmFKF1 gene editing positive plants were obtained and verified by using the specific primers of cas9. The target site amplification and sequencing analysis was used to determine whether the T1 transgenic lines had mutation at the expected target site of ZmFKF1 and the type of mutation, and screened the homozygous lines of ZmFKF1 site mutation. After obtaining the above materials, the flowering phenotypes of these materials were statistically analyzed with wild type as control. At the same time, qRT-PCR was used to detect the expression of key genes that related to flowering pathway in the above materials and further verify the phenotype. 【Result】The target sequence was designed on exon1 of ZmFKF1 to construct gene editing expression vector. The transgenic lines were obtained by genetic transformation realized site directed mutagenesis of ZmFKF1. A total of 18 T0 generation ZmFKF1 gene-edited lines were obtained, and 6 of them were generated mutation on the exon1 with two different mutations types, including single base insertion and multiple bases deletion. The phenotype analysis shown that the flowering time of three T2 generation ZmFKF1 homozygous lines was delayed compared with wild type B104. Furthermore, the expression levels of three related flowering pathway genes (ZmGI, conz1 and ZmZCN8) in the mutants were significantly lower than those in the wild type B104 (P＜0.05), which was consistent with the late flowering phenotype of the mutants. 【Conclusion】CRISPR/Cas9 technology can be used to edit ZmFKF1 to obtain gene editing mutants, and the flowering time of these mutants is significantly delayed compared to wild type.

Key words: CRISPR/Cas9, gene editing, maize, ZmFKF1, flowering time

Table 1

Cloning and identification primers used in this study"

引物名称 Primer name 引物序列 Primer sequence (5′-3′) 用途 Usage
ZmFKF1-F CACCATGTTTGACGACGCGGGGGC ZmFKF1克隆
Cloning of ZmFKF1 gene
ZmFKF1-R GACTTTGCCATGCGAATCTACAATGC
FKF1-U6b-F GTTGAGGTGGACGCGGAGCCGGGA 载体构建及鉴定
Vector construction and verification
FKF1-U6b-R AAACTCCCGGCTCCGCGTCCACCT
U-F CTCCGTTTTACCTGTGGAATCG
gR-R CGGAGGAAAATTCCATCCAC
Pps-R TTCAGAggtctcTaccgACTAGTCACGCGTATGGAATCGGCAGCAAA
Pgs-L AGCGTGggtctcGctcgACGCGTATCCATCCACTCCAAGC
SP-L1 GCGGTGTCATCTATGTTACTAG
SP-R TGCAATAACTTCGTATAGGCT
Cas9-F CTGACGCTAACCTCGACAAG 鉴定Cas9
PCR detection for Cas9
Cas9-R CCGATCTAGTAACATAGATGACACC
ZmCri-F CCCTACCTGGCAGCAGAATC 靶点检测及测序引物
Target detection and sequencing primer
ZmCri-R AGCAGCCCAAATCTGTCAGTC
ZmUbi-F GTCTCCGTGGTGGTCAGTAAGT qRT-PCR检测引物
Primers used for qRT-PCR assay
ZmUbi-R GACACGAACAGCAGATACTTTGAC
ZmGI-F ACTTTCCCAACCAACGC
ZmGI-R GGGCAGCTACAACAATTTCA
conz1-F ATCGTCAATGGAGGTGGGTAT
conz1-R GACCCGACGAGAACAGGC
ZmZCN8-F ACTTGCACTGGATGGTGACAGATA
ZmZCN8-R CGGCCTCTCATAAAATATTAGCTCTT

Fig. 1

Target site of the gRNA in the ZmFKF1 gene, phylogenetic tree analysis of FKF1 amino acid sequences in nine species and the diagram of T-DNA region of pYLCRISPR/Cas9-ZmFKF1-T1 plasmid A: Position of the target in ZmFKF1 gene locus; B: Phylogenetic tree analysis of FKF1 amino acid sequences in 9 species; C: The diagram of T-DNA region of pYLCRISPR/Cas9-ZmFKF1-T1 plasmid"

Fig. 2

Transformation process of immature embryo of B104 A: Immature embryo of B104; B: Primary callus; C: Resistant callus; D: Resistant regeneration buds; E: Resistant regeneration plantlets"

Fig. 3

Identification and sequencing analysis of ZmFKF1 editing lines A: Identification of positive ZmFKF1 gene editing lines; B: Sequencing results of ZmFKF1 editing homozygote"

Table 2

Mutation analysis on the exon1 of ZmFKF1 gene"

株系 Line 序列 Sequence (5′-3′) 突变类型 Mutation type 是否纯合 Homozygous or not
B104 AGGTGGACGCGGAGCCGGGATGG --- ---
C1-6 AGGTGGACGCGGAGCTATGG +1（T）,-4（CGGG） 否 No
C2-12 AGGTGGACGCGGAGCCGAGGATGG +1（A） 是 Yes
C3-3 AGGTGGACGCGGAGCCG -9（GGATGGCCG） 否 No
C5-7 AGGTGGACGCGGAGCCGGGATGG -20（AGGTGGACGCGGAGCCGGGA） 否 No
C6-2 AGGTGGACGCGGAGCCATGG -3（GGG） 是 Yes
C7-1 AGGTGGACGCGGAGCCG -6（GGGATG） 是 Yes

Fig. 4

Prediction of ZmFKF1 protein domain and analysis of ZmFKF1 coding amino acid sequence in mutants A: Prediction results of ZmFKF1 protein domain; B: Amino acid sequence prediction analysis and comparison results of ZmFKF1 in ZmFKF1gene editing mutants"

Fig. 5

Phenotype and flowering related genes expression analysis of ZmFKF1 editing lines A: The phenotype of C7-1 compared to ‘B104’ at 67 days after sowing; B: The phenotype of C7-1 compared to ‘B104’ at 70 days after sowing; C: Expression analysis of flowering related genes in different genotype lines. ZmUbi primers were used for PCR as an internal control, the result of relative genes expression was calculated by 2-ΔΔCT method"

Table 3

Statistical results of flowering time in different genotype lines"

基因型
Genotype		 抽雄时间
Male phase		 散粉时间
Pollen phase		 吐丝时间
Female phase
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*
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