Functional Differentiation of BnVTE4 Gene Homologous Copies in α-Tocopherol Biosynthesis Revealed by CRISPR/Cas9 Editing

Tocopherols are essential nutrients for human health known as vitamin E. Vitamin E deficiency can have a profound effect on human health, including the central nervous system and cardiovascular and immune protection. Multiple enzymatic steps are involved in the conversion between different forms of tocopherols. Among them, γ-tocopherol methyltransferase encoded by gene VTE4 catalyzes the conversion of γ- to α-tocopherol or δ- to β-tocopherol isoforms. However, the gene copies and their functional contribution of VTE4 homologs in Brassica napus were not elucidated. To this end, different mutation combinations of four putative BnVTE4 homologous copies were generated by using CRISPR/Cas9 genome editing technology. Editing of those BnVTE4 homologs led to a significant change of the α-tocopherol content and the ratio between α- and γ-tocopherol compared with wide-type control. Analysis of the different combinations of BnVTE4-edited homologs revealed that the contribution of the BnVTE4 individual gene displayed obvious functional differentiation in α-tocopherol biosynthesis. Their contribution could be in order of VTE4.C02-2 (BnaC02G0331100ZS) > VTE4.A02-1 (BnaA02G0247300ZS) > VTE4.A02-2 (BnaA02G0154300ZS). Moreover, the VTE4.A02-1 and VTE4.A02-2 copies might have severe functional redundancies in α-tocopherol biosynthesis. Overall, this study systemically studied the different effects of BnVTE4 homologs, which provided a theoretical basis for breeding high α-tocopherol content oilseed rape.