Identification of Developing-related Gene <i>ZmCen</i> Using CRISPR/Cas9 in Maize

doi:10.13523/j.cb.2008119

China Biotechnology

2020, Vol. 40

Issue (12): 49-57 DOI: 10.13523/j.cb.2008119

Identification of Developing-related Gene ZmCen Using CRISPR/Cas9 in Maize

LEI Hai-ying¹,ZHAO Qing-song¹,BAI Feng-lin¹,SONG Hui-fang¹,WANG Zhi-jun^2,^**(

)

1 Faculty of Biology Sciences and Technology, Changzhi University, Changzhi 046011, China
2 Department of Chemistry, Changzhi University, Changzhi 046011, China

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Abstract

Objective: To construct a ZmCen gene expressing vector using CRISPR/Cas9 system and analyze its effect on growth and development in maize after transformation. Methods: A sgRNA in the first exon of ZmCen gene was designed targeting it. The sgRNA was inserted into the pOMS01-Cas9-ZmCen-sgRNA vector, and its transgenic lines B104 were obtained via Agrobacterium-mediated transformation, subcultured, induced and differentiated into seedlings. The T₀ and T₁ generations genomic DNA was amplified and analyzed, and the plants were phenotype comparison screening. Results: The ZmCen expressing vector was successfully constructed. The genomic DNA of T₀ and T₁ generations was detected by PCR and sequenced. The mutagenesis frequency for ZmCen was 20.13% and 64.52% in T₀ and T₁ transgenic lines, respectively. The frequency of homozygous deletion mutation was 5% in T₁ transgenic lines. Sequence analysis showed that base substitutions, insertions, or deletions occurred near the editing target of the ZmCen gene. Compared with the wild-type phenotype, it was found that the T₁ generation plants of ZmCen mutant showed incomplete male inflorescence phenotype, while the male inflorescence of homozygous mutant plants was sterility. Conclusion: The ZmCen gene was succeed editing by CRISPR/Cas9 technical in maize, and the successful construction of ZmCen mutants lays a foundation for the related-genes study of maize male organ development.

Key words： CRISPR/Cas9 Maize ZmCen male inflorescence

Received: 11 August 2020 Published: 14 January 2021

ZTFLH:

Q789

Corresponding Authors: Zhi-jun WANG E-mail: czxywzj@163.com

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	Hai-ying LEI
	Qing-song ZHAO
	Feng-lin BAI
	Hui-fang SONG
	Zhi-jun WANG

Cite this article:

LEI Hai-ying,ZHAO Qing-song,BAI Feng-lin,SONG Hui-fang,WANG Zhi-jun. Identification of Developing-related Gene ZmCen Using CRISPR/Cas9 in Maize. China Biotechnology, 2020, 40(12): 49-57.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2008119 OR https://manu60.magtech.com.cn/biotech/Y2020/V40/I12/49

Table 1 The PCR primers sequences used in this study

Table 2 Expression vector construction system

Fig.1 Schematic diagram of ZmCen gene structure and the result of pOMS01-Cas9-ZmCen-gRNArecombination expression vector (a) The gene structure of ZmCen and design of the sgRNA . The target site of ZmCen-sgRNA in exon 1. The PAM motif is the TGG,which is located in the end of nucleotide sequence LB: Vector left border; RB: Vector right border; Bar: The resistance gene of herbicide; Ubi promoter: Ubiquitin promoter; OsU3 promoter: Rice U3 promoter; 35S promoter: Cauliflower mosaic virus (CaMV) 35S promoter; Nos terminator: Agrobacterium tumefaciens nopaline synthase gene (Nos) terminator; gRNA: Guide RNA; Cas9: The gene of Cas9 (b)Identification of the recombinant expression vector pOMS01-Cas9-ZmCen-gRNA M: Marker; 1 and 2: The PCR result of the positive clones

Fig.2 Partial T₀ generation test strips and the agarose gel electrophoresis results of the PCR amplification (a) The result of test strips +: Positive; -: Negative (b) Agarose gel electrophoresis of the editing target sequences amplification products M: Standard molecular weight maker; 1-17: T₀ transgenic lines

Fig.3 Partial comparison result of the editing target sequences between T₀ and T₁ transgenic plants

Tab.3 sgRNA gene editing mutation type detection

Fig.4 The PCR agarose gel electrophoresis result of Cas9 gene in T₀ and T₁ transgenic generations (a) The result of T₀ transgenic generations (b) The result of T₁ transgenic generations +:Positive control; -:Negative control;M: Standard molecular weight maker

Fig.5 Comparison of male inflorescence phenotype, anther and pollens between wild type and T₁ mutation lines (a), (d),(f)and(h) were wild type; (b), (c), (e), (g) and (i) were T₁ mutation lines


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