Abstract：
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 Brassica napus (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 CYP704B genes has been used to create new male sterile lines in many crops. In the present study, a total of 687 BnCYP450 genes were identified in Brassica napus cultivar \"Zhongshuang 11\" (ZS11), which has nearly 2.8-fold as many CYP450 members as in Arabidopsis thaliana. It is rationally estimated since Brassica napus is a tetraploid oil plant with a larger genome compared with Arabidopsis thaliana. The BnCYP450 genes were divided into 47 subfamilies and clustered into nine clans. Phylogenetic relationship analysis reveals that CYP86 clan consists of four subfamilies and 109 BnCYP450s. Members of CYP86 clan genes display specific expression profiles in different tissues and in response to ABA and abiotic stresses. Two BnCYP450s within the CYP704 subfamily from CYP86 clan, BnCYP704B1a and BnCYP704B1b, display high similarity to MS26 (Male Sterility 26, also known as CYP704B1). These two BnCYP704B1 genes were specifically expressed in young buds. We then simultaneously knocked-out these two BnCYP704B1 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 Brassica napus. This study provides a systemic view of BnCYP450s 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 GMS controlling genes.
摘要：
细胞色素P450（CYP450）单加氧酶超家族，参与许多初级和次级代谢物的生物合成途径，在植物生长发育中起着重要作用。然而,关于甘蓝型油菜CYP450（BnCYP450）的系统信息以前未被发现，其生物学意义远未被理解。家族成员86CYP450,如CYP704B，是植物雄性繁殖中花粉壁形成所必需的，CYP704B基因的靶向诱变已被用于在许多作物中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)中共鉴定出687个BnCYP450基因，CYP450成员的数量几乎是拟南芥的2.8倍。这是合理估计的，因为甘蓝型油菜是一种四倍体油料植物，与拟南芥相比具有更大的基因组。BnCYP450基因分为47个亚家族，并聚集为9个氏族。系统发育关系分析表明，CYP86家族由四个亚家族和109个BnCYP450组成。CYP86氏族基因的成员在不同组织中以及对ABA和非生物胁迫的响应中显示出特定的表达谱。来自CYP86家族的CYP704亚家族中的两个BnCYP450，BnCYP704B1a和BnCYP704B1b，显示与MS26(雄性不育26,也称为CYP704B1)的高度相似性。这两个BnCYP704B1基因在幼芽中特异性表达。然后，我们通过成簇的规则间隔的短回文重复序列/CRISPR相关蛋白9（CRISPR/Cas9）基因组工程系统同时敲除了这两个BnCYP704B1基因。编辑过的植物显示出无花粉，成熟花药的不育表型，表明我们成功复制了基因雄性不育(GMS，甘蓝型油菜中也称为核雄性不育）系。这项研究提供了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GMS控制基因在油菜中快速生成GMS的商业实用性。
