A previous work (see BioProject PRJNA623495) investigated the contribution of the introgression of an over-expressing copy of MYB28 from Brassica villosa into a Brassica oleracea var italica background, where the genes involved in glucosinolate biosynthesis and sulphur metabolism were found to be up-regulated.
More...A previous work (see BioProject PRJNA623495) investigated the contribution of the introgression of an over-expressing copy of MYB28 from Brassica villosa into a Brassica oleracea var italica background, where the genes involved in glucosinolate biosynthesis and sulphur metabolism were found to be up-regulated. To confirm this role of MYB28, knockout lines were generated in Brassica oleracea using CRISPR-Cas9 mediated gene editing technology and were grown in the field. These mutants also served to investigate whether the reverse effect on the sulphur metabolome genotype of the MYB28 over-expressing high glucoraphanin Inbred broccoli would be found when MYB28 function is impaired.
The T2 generation of the myb28 CRISPR mutant lines were found to have two types of mutation in the C2 copy of MYB28 segregating within the population. One of which was a single base pair insertion, a thymine, which led to a frameshift mutation in which a premature stop codon was introduced. Lines with this mutation were characterised as mutant 1. In addition, some of the lines contained a mutation appearing at the same point in the protein sequence in which a three base pair deletion occurs, leading to the deletion a single amino acid, a leucine, in the resulting protein. Lines with this mutation were characterised as mutant 2. Both lines have a 562 base pair deletion in the C9 copy of MYB28, which likely renders the resulting protein non-functional
The Brassica oleracea mutants described in this experiment were also used in and the first DEFRA regulated and approved CRISPR field trial in the UK to require compliance with the 2001/18 EU GMO directive (see https://www.gov.uk/government/publications/genetically-modified-organisms-john-innes-centre-19r5201). Less...