机构地区：[1]Institute of Biochemistry and Genetics, Subdivision of the Ufa Federal Research Centre of the Russian Academy of Sciences, Ufa, Russia [2]Federal Centre of Agriculture Research of South-East Region, Saratov, Russia [3]Kazan Federal University, Institute of Fundamental Medicine and Biology, Kazan, Russia [4]Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany
出 处：《American Journal of Plant Sciences》2021年第8期1276-1287,共12页美国植物学期刊（英文）
摘 要：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 immaturSorghum (<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
关 键 词：Sorghum bicolor (L.) Moench CRISPR/Cas Genome Editing α-Kafirin γ-Kafirin Genetic Engineering Grain Quality
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