Functional analysis and mutagenesis of GDSL genes for breeding oilseed rape (B. napus) with higher oil content
Seed oil content is determined by the balance between seed fatty acids synthesis and degradation. To date, many attempts have been made to increase oil content in rapeseed via improving seed fatty acid biosynthesis. On the other hand, it has been reported that after an increase during the first 35 days after pollination, rapeseed oil content is declining to a small extent at the late stage of seed development until maturation, indicating that degradation processes might play a role (Fowler and Downey 1970; Kelly et al. 2013). In Arabidopsis, five Seed Fatty Acid Reducer (SFAR) genes have been identified, which belong to the GDSL lipase/esterase family and have been shown to be involved in the seed fatty acid reduction in mature seeds (Chen et al. 2012).
Due to the close evolutionary relationship of Arabidopsis and Brassica napus, we started to investigate the effect of corresponding homologous gene copies on the oil content in rapeseed. In this project, we follow two knock-out approaches using classical TILLING in an existing EMS (ethylmethanesulfonate) mutated winter rapeseed population and the CRISPR/Cas9 technique.
The objectives of this project are to identify missense and nonsense mutations within BnSFAR genes by TILLING and produce homozygous mutant lines. Single mutants are crossed to obtain double mutants and backcrosses are performed to reduce the mutational load of the mutant lines. For comparison, the CRISPR/Cas9 system is used to obtain knock-out mutants without mutation background and for simultaneous knock-out of more than one candidate gene. Mutants will be analyzed phenotypically for oil content, fatty acid profile and lipase activity in the developing seeds. In addition, expression profiles and seedling performance will be monitored.
By in silico analysis, for 5 known Arabidopsis SFAR genes 12 homologous genes were identified in B. napus. The coding regions were varying from 1053 bp to 1308 bp, encoding for 351 to 436 amino acids. Paralog copies were classified into subgroups in alignments using structural homologies on DNA level as well as in the five conserved protein domains of the GDSL type BnSFAR proteins.
Eight BnSFAR genes were chosen for TILLING and for CRISPR/Cas9 genome editing experiments, according to their highest gene expression in developing seeds. The mutation screening is currently on the way.
Professor Lixi Jiang, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China
Norddeutsche Pflanzenzucht Innovation GmBH, Hohenlieth
Harloff HJ, Lemcke S, Mittasch J, Frolov A, Wu JG, Dreyer F, Leckband G, Jung C (2012) A mutation screening platform for rapeseed (Brassica napus L.) and the detection of sinapine biosynthesis mutants. Theor Appl Genet 124: 957-969
Financial Support (three years)
The project is a joint Sino-German research project.
It is funded by the Sino-German Center (GZ1099) and the Deutsche Forschungsgemeinschaft under grant No. DFG JU 205/25-1
Updated: 16.09.2016 Responsible for this webpage: Nirosha Lakmali