【摘要】 绿色革命中“理想株型”概念的提出为玉米(Zea mays)株型设计育种提供了新思路。过长过宽的叶片由于披垂会导致产量下降，而卷叶能使叶片保持直立而不披垂，增大群体的受光面积，有效提高光合效率。本研究以玉米经典自然突变体cr1 (Crinkly Leaves 1)(表现为叶片褶皱,植株矮小)为材料，对该突变体进行细胞学分析、图位克隆、基因编辑和表达分析。结果表明，该突变体叶片的平铺细胞发生形变且排列不规则，目标基因ZmCYP90D1定位在3号染色体短臂末端0.9 Mb的区间内。对区间内29个编码基因进行功能分析和外显子测序后，发现在cr1中ZmCYP90D1的第3个外显子缺失。进一步基因组测序发现，在cr1中第3个外显子末端的剪接位点处插入了1.2 kb的转座子，导致ZmCYP90D1的mRNA被错误剪接，丢失第3个外显子。ZmCYP90D1 (Zm00001d039453)基因编码细胞色素单加氧酶，参与油菜素内酯的生物合成，定位在内质网。基因编辑产生的5个纯合敲除系都表现出叶片褶皱，植株矮小的表型。对其中2个敲除系ko1和ko2进行分析，确认ZmCYP90D1即是cr1的目标基因。RNA-seq结果表明，差异表达基因多富集在细胞分裂和分化、细胞壁的形成和修饰、激素调节的响应、脂肪酸合成代谢等生物学过程。本研究为深入解析油菜素内酯合成途径调控玉米叶片发育的分子机制提供理论基础。更多还原

【Abstract】 The concept of ’optimum plant types’ in the Green Revolution provides a new idea for the design and breeding of maize(Zea mays) plant type. Over-long and over-wide leaves will lead to decreased yield due to draping, while broad leaves can keep the leaves upright without draping, increase the light receiving area of the population, and effectively improve the photosynthetic efficiency. The classic maize(Zea mays) mutant Crinkly Leaves 1(cr1) showed crinkled leaves and dwarf plants. The following results were obtained after cytological analysis, mapping cloning, gene editing and expression analysis of the mutant. The pavement cells of the mutant’s leaves were deformed and irregularly arranged, and the mapping cloning results showed that the target gene ZmCYP90D1 was located within 0.9 Mb at the end of the short arm in chromosome 3. After functional analysis and exon sequencing of 29 coding genes in the region, it was found that the third exon of ZmCYP90D1 was missing in cr1 mutant. Further genome sequencing revealed that a 1.2 kb transposon was inserted at the splicing site at the end of the third exon of the cr1, resulting in the missplicing of the mRNA of ZmCYP90D1 and the loss of the third exon. The ZmCYP90D1(Zm00001d039453) gene encoded cytochrome monooxase, which was involved in the biosynthesis of brassinolactone and located in the endoplasmic reticulum. Gene editing produced 5 homozygous knockout lines, all of which showed crinkly leaf and short plant phenotype. Two of the knockout lines, ko1 and ko2, were analyzed, and ZmCYP90D1 was confirmed as the target gene of cr1. RNA-seq showed differentially expressed genes were mainly concentrated in biological processes such as cell division and differentiation, cell wall formation and modification, hormone regulation response, fatty acid anabolism and so on. The identification of cr1 laid a foundation for further analysis of the molecular mechanism of brassinolide synthesis pathway regulating maize leaf development.更多还原