PSH121 (NCBI: txid2338066) ( Figure 1(a)) was used as the basic vector for the introduction of target-specific sequences of kafirin-encoding genes uponįigure 1. Further, the Phosphinothricin phosphotransferase ( Bar) gene of Streptomyces hygroscopicus equipped with an intron to prevent agrobacterial expression and driven by the maize UBI1 promoter was used as plant selectable marker. Accordingly, major features of the constructs generated in the present study include the rice U3 promoter and the maize POLYUBIQUITIN 1 ( UBI1) promoter to drive gRNA (guide RNA) and cas9 expression, respectively. The aim of this work was to create highly efficient vectors and agrobacterial clones containing these vectors to mutate the α- and γ- KAFIRIN genes of sorghum. Previous studies have revealed a multitude of aspects that have to be considered when generating transformation vectors for plant genome editing using Cas endonucleases. Recently published work on the induction of mutations in the α-kafirin nucleotide sequence has shown the potential of Cas endonuclease technology to improve the nutritional value of sorghum grain. As a further consequence, the proteome of caryopses may be rebalanced via enhanced synthesis of other proteins, including those with a higher content of essential amino acids such as lysine. The reduction of kafirin synthesis induces the changes in the ultrastructure of endosperm protein bodies and increases their digestibility by proteases. The targeted induction of mutations in genes encoding different classes of kafirins, including gene knockouts, using genome editing bears the potential to significantly improving the digestibility of proteins in sorghum grain and increase its nutritional value. Ĭas endonuclease technology offers to solve this problem. The poor digestibility of kafirins, in turn, reduces the access of amylolytic enzymes to starch granules and reduces the digestibility of starch and the nutritional value of sorghum grain. However, compared to other cereals, sorghum grain has a lower nutritional value, the main reason for which is the resistance of its grain storage proteins (kafirins) to protease digestion. Sorghum grains do not contain gluten and can serve as a source of protein for people with gluten intolerances, which must follow a gluten-free diet. Due to global warming of climate, the importance of this crop is expected to grow steadily. Sorghum ( Sorghum bicolor (L.) Moench) is one of the most important drought-tolerant cereal crops in the arid regions of the Earth. To date, this technology allowed to modify many agronomically important traits in major cultivated crops, such as corn, rice, wheat, potatoes, soybeans, sugarcane, etc. Unlike traditional strategies and breeding methods, Cas endonuclease technology provides a fast path to the creation of modified genotypes through site-directed mutagenesis or precise editing of the nucleotide sequences of respective genes.
SOLID V DOTTED LINE IN MAP SNAPGENE VIEWER SERIES
Transferred by electroporation into the Agrobacterium tumefaciens strain AGL0.Ĭurrently, this vector series is used for stable transformation of sorghumĪmong the many biotechnological approaches for improving the properties of agricultural plants, genome editing has the potential to play a key role. The correct assembly ofīinary vectors was confirmed by restriction analysis using the MluI and SfiI cleavage sites. Sites into the compatible binary vector B479p7oUZm-LH. Subcloning of the resulting constructs was performed using the SfiI restriction Validation of the cloning procedure was performed by DNA sequencing. The insertion of respective sequences in the generic vector pSH121 was Two most suitable targets were chosen for α-KAFIRIN ( k 1 C5) and two Theĭesign of the gRNAs was conducted using the online tools CRISPROR andĬHOPCHOP. Sequences) were selected for the introduction of mutations into the α- and γ-KAFIRIN genes of sorg hum. In this study, genomic target motifs (23 bp The employment of genome editing may allow to solve this problemīy introducing mutations into the nucleotide sequences of the α- and γ-kafirin genes. Partially or completely suppressed synthesis or altered amino acid composition Improve the nutritional value of sorghum grain is to obtain mutants with Proteins (kafirins) to protease digestion. Nutritional value, which is caused, inter alia, by the resistance of its seed storage However, unlike other cereals, sorghum grain has a lower Sorghum ( Sorghum bicolor (L.) Moench) is one of the world’s leadingĬereal crops in agricultural production, which has a special importance in theĪrid regions.
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