2021年1月27日，中国农业科学院蔬菜花卉研究所甘蓝青花菜团队在基因定位经典期刊Theoretical and Applied Genetics（2023 IF=5.4）上发表了标题为Identification and validation of an ECERIFERUM2‑ LIKE gene controlling cuticular wax biosynthesis in cabbage (Brassica oleracea L. var. capitata L.)的研究论文，定位并鉴定了蜡质缺失基因BoCER2。论文的第一作者为季家磊博士和曹文雪博士，通讯作者为吕红豪研究员。

TL28-1是一个甘蓝蜡质缺失突变体，通过构建群体进行遗传分析和基因定位，成功定位到导致其蜡质缺失的基因BoCER2。遗传分析表明蜡质缺失性状为单基因隐性遗传，精细定位表明蜡质缺失位点wdtl28基因位于1号染色体的BOL01-20和BoL01-24标记之间的80Kb区间。根据甘蓝基因组注释，确定ECERIFERUM2(CER2-like)基因BoCER2为候选基因。系统发育分析表明BoCER2和其他类似CER2的基因在BAHD酰基转移酶超家族中形成了一个分支。组织表达特异性分析BoCER2在茎、叶、花和角果等组织中均有表达，但在甘蓝根中未检测到。亚细胞定位表明BoCER2蛋白在内质网发挥功能。进一步的序列分析表明，TL28-1的BoCER2编码序列存在单核苷酸突变(G到A)，导致终止密码子(TGA)，从而提前终止翻译。连锁分析表明，BoCER2纯合突变与蜡质缺乏症共分离。此外，互补试验表明，野生型BoCER2可以弥补TL28-1的蜡质缺失。

Fig. 1 Characterization of the TL28-1 mutant. a Plant appearance of wild-type (WT). b Plant appearance of the mutant TL28-1.c Microstructure of WT leaf surface. d Microstructure of TL28-1 leaf surface. e Total leaf
wax load of WT and TL28-1 measured as average values for three biological replicates. f. Wax composition of WT and TL28-1 leaves, measured as average values for three biological replicates

Fig. 2 Map-based cloning of wdtl28. The schematic diagram depicts the exon (solid black box) and intron (black line) of wdtl28. Nucleotide sequence depicting the point mutation in TL28-1. Asterisk indicates the conserved nucleotide site in CER2-homologous genes from different species

Fig. 3 Linkage analysis and complementation test. a Linkage analysis of different cabbage lines using a KASP marker within BoCER2. Red dots indicate the recessive glossy plants with A/A (T/T) genotype from the F2 population; blue dots indicate glaucous cabbage lines with G/G (C/C) genotype. b Glyphosate-resistant buds obtained after genetic transformation. c A positive transgenic plant. d Microstructure of the transgenic plant leaf surface (color figure online)

Fig. 4 Phylogenetic analysis of the CER2-LIKEs and the BAHD acyltransferases. The phylogenetic tree was constructed using FastTree based on the W AG + CAT model. The clade with a sky blue background was designated as “CER2-LIKE clade”. Accession numbers: AtCER2, At4g24510; OsCER2, Os04g0611200; Zm_CER2-LIKE, NP_001309865.1 (NCBI); Rc_CER2-LIKE, XP_002514050.1 (NCBI); Md_CER2-LIKE, XP_008338959.1 (NCBI) (color figure online)

Fig. 5 The spatial expression pattern and subcellular localization analysis of BoCER2. a qPCR analysis of BoCER2 expression pattern in different tissues of wild-type. b Fluorescence signals of BoCER2 fused with GFP. c Endoplasmic reticulum marker. d Fluorescence signals of chloroplast. e Bright-field image. f Superposition image of bright field and fluorescence