TITLE:
Binary Vector Construction for Site-Directed Mutagenesis of Kafirin Genes in Sorghum
AUTHORS:
Grigoriy A. Gerashchenkov, Lev A. Elkonin, Kirill G. Gerashchenkov, Natalia A. Rozhnova, Stefan Hiekel, Jochen Kumlehn, Alexey V. Chemeris
KEYWORDS:
Sorghum bicolor (L.) Moench, CRISPR/Cas, Genome Editing, α-Kafirin, γ-Kafirin, Genetic Engineering, Grain Quality
JOURNAL NAME:
American Journal of Plant Sciences,
Vol.12 No.8,
August
30,
2021
ABSTRACT: Sorghum (Sorghum bicolor (L.) Moench) is one of the world’s leading
cereal crops in agricultural production, which has a special importance in the
arid regions. However, unlike other cereals, sorghum grain has a lower
nutritional value, which is caused, inter alia, by the resistance of its seed storage
proteins (kafirins) to protease digestion. One of the effective approaches to
improve the nutritional value of sorghum grain is to obtain mutants with
partially or completely suppressed synthesis or altered amino acid composition
of kafirins. The employment of genome editing may allow to solve this problem
by introducing mutations into the nucleotide sequences of the α- and γ-kafirin genes. In this study, genomic target motifs (23 bp
sequences) were selected for the introduction of mutations into the α- and γ-KAFIRIN genes of sorghum. The
design of the gRNAs was conducted using the online tools CRISPROR and
CHOPCHOP. Two most suitable targets were chosen for α-KAFIRIN (k1C5) and two
for γ-KAFIRIN (gKAF1)
genes. The insertion of respective sequences in the generic vector pSH121 was
performed at the BsaI (Eco31I)
sites. Validation of the cloning procedure was performed by DNA sequencing.
Subcloning of the resulting constructs was performed using the SfiI restriction
sites into the compatible binary vector B479p7oUZm-LH. The correct assembly of
binary vectors was confirmed by restriction analysis using the MluI and SfiI cleavage sites. The four vectors created (1C - 4C) were
transferred by electroporation into the Agrobacterium tumefaciens strain AGL0.
Currently, this vector series is used for stable transformation of sorghum
using immature embryo explants.