SITE-directed mutagenesis for producing grain sorgum mutants with improved kafirine digestibility

Lev A. Elkonin; Grigoryi A. Gerashchenkov; Natalie V. Borisenko; Odissey A. Kenzhegulov; Saule Kh. Sarsenova; Valery M. Panin; Natalia A. Rozhnova

doi:10.17816/ecogen567897

SITE-directed mutagenesis for producing grain sorgum mutants with improved kafirine digestibility

Authors: Elkonin L.A.¹, Gerashchenkov G.A.², Borisenko N.V.¹, Kenzhegulov O.A.¹, Sarsenova S.K.¹, Panin V.M.¹, Rozhnova N.A.²
Affiliations:
1. Federal Centre of Agriculture Research of South-East Region
2. Institute of Biochemistry and Genetics — Subdivision of the Ufa Federal Research Centre of the Russian Academy of Sciences
Issue: Vol 21 (2023): Спецвыпуск
Pages: 48-49
Section: Genetically modified organism. The Нistory, Achivements, Social and Environmental Riscs
URL: https://journals.eco-vector.com/ecolgenet/article/view/567897
DOI: https://doi.org/10.17816/ecogen567897
ID: 567897

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Abstract

The use of genome editing technologies opens wide opportunities for the targeted mutagenesis in important agricultural crops. In the context of global warming, sorghum, an important drought- and heat-tolerant crop is of particular importance. However, compared to other cereals, sorghum grain has a lower nutritional value, due to the resistance of its storage proteins (kafirins) to proteolytic digestion. A decrease in the synthesis of kafirins as a result of mutations or the expression of the RNAi genetic constructs modifies the ultrastructure of protein bodies and improves their digestibility by proteases. To obtain mutants with improved protein digestibility, we created four binary vectors for site-directed mutagenesis of the k1C5 and gKAF1 genes encoding α- and γ-kafirin, respectively. Each of these vectors contained the cas9 endonuclease gene and a guide RNA targeted the nucleotide sequences encoding the kafirin signal polypeptides. By means of agrobacterial transformation, the created vectors were introduced into the genome of the grain sorghum cv. Avans. 14 transgenic plants were regenerated. Sequencing of 5 regenerants obtained using a vector for the k1C5 mutagenesis revealed 3 plants with mutations. The offspring of these mutants had a higher digestibility of grain proteins in vitro (86–92%) compared to the initial cv. Avans (63–67%). Notably, the T₁ plants lacked the cas9 gene and the bar marker gene, which indicates the production of mutants with the edited k1C5 gene sequence, which lack the genetic construct that induced this mutation. Two mutants with mutations in the gKAF1 sequence were obtained. Thus, using the CRISPR/Cas technology, we have obtained mutants with improved digestibility of kafirins, which can be used in practical sorghum breeding.