Wang Zhilai, Zhang Yanfeng, Song Min, Tang Xiuhua, Huang Shuhua, Linhu Bin, Jin Ping, Guo Weike, Li Fang, Xing Liwen, An Ran, Zhou Xiaona, Hao Wenfang, Mu Jianxin, Xie Changgen
State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling 712100, China.
Hybrid Rapeseed Research Centre of Shaanxi Province, Yangling 712100, China.
Plants (Basel). 2023 Jan 12;12(2):365. doi: 10.3390/plants12020365.
The cytochrome P450 (CYP450) monooxygenase superfamily, which is involved in the biosynthesis pathways of many primary and secondary metabolites, plays prominent roles in plant growth and development. However, systemic information about CYP450s in (BnCYP450) was previously undiscovered and their biological significance are far from understood. Members of clan 86 CYP450s, such as CYP704Bs, are essential for the formation of pollen exine in plant male reproduction, and the targeted mutagenesis of genes has been used to create new male sterile lines in many crops. In the present study, a total of 687 genes were identified in cultivar "Zhongshuang 11" (ZS11), which has nearly 2.8-fold as many members as in . It is rationally estimated since is a tetraploid oil plant with a larger genome compared with . The genes were divided into 47 subfamilies and clustered into nine clans. Phylogenetic relationship analysis reveals that clan consists of four subfamilies and 109 . Members of clan genes display specific expression profiles in different tissues and in response to ABA and abiotic stresses. Two within the subfamily from clan, and , display high similarity to (, also known as ). These two genes were specifically expressed in young buds. We then simultaneously knocked-out these two genes through a clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome engineering system. The edited plants displayed a pollenless, sterile phenotype in mature anthers, suggesting that we successfully reproduced genic male sterility (GMS, also known as nuclear male sterility) lines in . This study provides a systemic view of and offers a strategy to facilitate the commercial utility of the CRISPR/Cas9 system for the rapid generation of GMS in rapeseed via knocking-out controlling genes.
细胞色素P450（CYP450）单加氧酶超家族参与许多初级和次级代谢产物的生物合成途径，在植物生长发育中发挥着重要作用。然而，此前尚未发现甘蓝型油菜（BnCYP450）中CYP450的系统信息，其生物学意义也远未明确。86族CYP450成员，如CYP704Bs，在植物雄性生殖中花粉外壁的形成至关重要，并且在许多作物中，对这些基因进行靶向诱变已被用于创建新的雄性不育系。在本研究中，在甘蓝型油菜品种“中双11”（ZS11）中总共鉴定出687个BnCYP450基因，其BnCYP450成员数量几乎是拟南芥的2.8倍。合理估计这是因为甘蓝型油菜是一种四倍体油料作物，与拟南芥相比基因组更大。这些BnCYP450基因被分为47个亚家族并聚类为9个族。系统发育关系分析表明，86族由四个亚家族和109个BnCYP450组成。86族基因成员在不同组织中以及对脱落酸（ABA）和非生物胁迫有特定的表达谱。来自86族71亚家族的两个BnCYP450，BnCYP71A13和BnCYP71A14，与AtCYP71A13（也称为AtKLUH）高度相似。这两个BnCYP450基因在幼芽中特异性表达。然后，我们通过成簇规律间隔短回文重复序列/CRISPR相关蛋白9（CRISPR/Cas9）基因组编辑系统同时敲除这两个BnCYP450基因。编辑后的植株在成熟花药中表现出无花粉的不育表型，这表明我们在甘蓝型油菜中成功创制了基因雄性不育（GMS，也称为核雄性不育）系。本研究提供了甘蓝型油菜BnCYP450的系统视图，并提供了一种策略，以促进CRISPR/Cas9系统通过敲除控制基因在油菜中快速产生GMS的商业应用。
