1 College of Agriculture, Northeast Agricultural University, Harbin 150030, China; 2 Suihua Branch of Heilongjiang Academy of Agricultural Sciences, Suihua 152001, China; 3 Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 10081, China; 4 Yi'an County Agricultural Technology Extension Center, Qiqihar 161507, China
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.
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