Sorghum (<i>Sorghum</i><span> <i>bicolor</i></span> (L.) Moench) is one of the world’s leading cereal crops in agricultural production, which has a special importance in the arid r...Sorghum (<i>Sorghum</i><span> <i>bicolor</i></span> (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 <i>α</i>- and <i>γ</i>-kafirin genes. In this study, genomic target motifs (23 bp sequences) were selected for the introduction of mutations into the <i>α-</i> and <i>γ-KAFIRIN</i> genes of sorg<span>hum. The design of the gRNAs was conducted using the online tools</span> CRISPROR and CHOPCHOP. <a name="_Hlk55317737"></a>Two most suitable targets were chosen for <i>α-KAFIRIN</i> (<i>k</i><span>1<i>C</i>5</span>) and two for <i>γ-KAFIRIN</i> (<i>gKAF</i><span>1</span>) genes. The insertion of respective sequences in the generic vector pSH121 was performed at the <i>BsaI</i> (<i>Eco</i><span>31<i>I</i></span>) sites. Validation of the cloning procedure was performed by DNA sequencing. Subcloning of the resulting constructs was performed using the <i>SfiI</i> restriction sites into the compatible binary vector B479p7oUZm-LH. The correct assembly of binary vectors was confirmed by restriction analysis using the <i>MluI</i> and <i>SfiI</i> cleavage sites. The four vectors created (1C</span><span style="font-family:""> </span><span style="font-family:"">-</span><span style="font-family:""> </span><span style="font-family:"">4C) were transferred by electroporation into the <i>Agrobacterium</i><span> <i>tumefaciens</i></span> strain AGL0. Currently, this vector series is used for stable transformation of sorghum using immatur展开更多
文摘Sorghum (<i>Sorghum</i><span> <i>bicolor</i></span> (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 <i>α</i>- and <i>γ</i>-kafirin genes. In this study, genomic target motifs (23 bp sequences) were selected for the introduction of mutations into the <i>α-</i> and <i>γ-KAFIRIN</i> genes of sorg<span>hum. The design of the gRNAs was conducted using the online tools</span> CRISPROR and CHOPCHOP. <a name="_Hlk55317737"></a>Two most suitable targets were chosen for <i>α-KAFIRIN</i> (<i>k</i><span>1<i>C</i>5</span>) and two for <i>γ-KAFIRIN</i> (<i>gKAF</i><span>1</span>) genes. The insertion of respective sequences in the generic vector pSH121 was performed at the <i>BsaI</i> (<i>Eco</i><span>31<i>I</i></span>) sites. Validation of the cloning procedure was performed by DNA sequencing. Subcloning of the resulting constructs was performed using the <i>SfiI</i> restriction sites into the compatible binary vector B479p7oUZm-LH. The correct assembly of binary vectors was confirmed by restriction analysis using the <i>MluI</i> and <i>SfiI</i> cleavage sites. The four vectors created (1C</span><span style="font-family:""> </span><span style="font-family:"">-</span><span style="font-family:""> </span><span style="font-family:"">4C) were transferred by electroporation into the <i>Agrobacterium</i><span> <i>tumefaciens</i></span> strain AGL0. Currently, this vector series is used for stable transformation of sorghum using immatur
