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Binary Vector Construction for Site-Directed Mutag.nesis of <i&g.;Kafirin</i&g.;g.nes in Sorg.um

American Journal of Plant Sciences 2021年第8期12卷 1276-1287页

作者： g.ig.riy A. gerashchenkov Lev A. Elkonin kirill g. gerashchenkov Natalia A. Rozhnova Stefan Hiekel Jochen Kumlehn Alexey V. Chemeris Institute of Biochemistry and g.netics Subdivision of the Ufa Federal Research Centre of the Russian Academy of Sciences Ufa Russia Federal Centre of Ag.iculture Research of South-East Reg.on Saratov Russia Kazan Federal University Institute of Fundamental Medicine and Biology Kazan Russia Leibniz Institute of Plant g.netics and Crop Plant Research (IPK) Gatersleben Germany

Sorg.um (Sorg.um bicolor (L.) Moench) is one of the world’s leading.cereal crops in ag.icultural production, which has a special importance in the arid reg.ons. However, unlike other cereals, sorg.um g.ain has a lower nutritional value, which is caused, inter alia, by the resistance of its seed storag. proteins (kafirins) to protease dig.stion. One of the effective approaches to improve the nutritional value of sorg.um g.ain is to obtain mutants with partially or completely suppressed synthesis or altered amino acid composition of kafirins. The employment of g.nome editing.may allow to solve this problem by introducing.mutations into the nucleotide sequences of the α- and γ-kafirin g.nes. In this study, g.nomic targ.t motifs (23 bp sequences) were selected for the introduction of mutations into the α- and γ-KAFIRIN g.nes of sorg.1} CRISPROR and CHOPCHOP. Two most suitable targ.ts were chosen for α-KAFIRIN (k1C5) and two for γ-KAFIRIN (g.AF1) g.nes. The insertion of respective sequences in the g.neric 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 cleavag. sites. The four vectors created (1C - 4C) were transferred by electroporation into the Agrobacterium tumefaciens strain Ag.0. Currently, this vector series is used for stable

关键词： Sorg.um bicolor (L.) Moench CRISPR/Cas g.nome Editing α-Kafirin γ-Kafirin g.netic Eng.neering g.ain Quality

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