利用CRISPR/Cas9鉴定玉米发育相关基因ZmCen*

目的: 利用CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) 系统构建玉米中心蛋白(Centrin)的表达载体,经转化后分析其对玉米生长发育的影响。方法: 针对ZmCen基因的第一个外显子设计sgRNA,将其连入pOMS01-Cas9-ZmCen-sgRNA表达载体,转化农杆菌GV3101后,侵染玉米自交系材料B104的愈伤组织,经继代、诱导、分化成苗,筛选出转基因后代。对T₀代和T₁代基因组DNA进行PCR验证、测序及表型分析。结果: 成功构建ZmCen的表达载体。侵染农杆菌后,PCR测序显示,T₀ 代和T₁ 代突变率分别为 20.13% 和 64.52%,其中T₁ 代的纯合缺失突变率为5%。序列分析表明,ZmCen基因的编辑靶点附近发生了碱基的替换、插入或缺失。经与野生型表型比对发现,ZmCen 突变体T₁代植株出现发育缓慢且雄花序不完全发育表型,纯合突变体植株雄花序则完全不发育。结论: 通过 CRISPR/Cas9技术成功地对玉米ZmCen基因进行了编辑,ZmCen突变体的获得为玉米雄性器官发育相关基因的研究奠定了基础。

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

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.