Sorghum(Sorghum bicolor(L.) Moench) is a major world crop that is a reliable source of fodder and food grain in arid regions. However, unlike other cereals, sorghum grain has low nutritional value, owing mainly to the...Sorghum(Sorghum bicolor(L.) Moench) is a major world crop that is a reliable source of fodder and food grain in arid regions. However, unlike other cereals, sorghum grain has low nutritional value, owing mainly to the resistance of its storage proteins(kafirins) to protease digestion. Changing the composition of kafirins or their primary structure may address this problem. To induce mutations in kafirin-encoding genes that were expected to disturb their accumulation in endosperm cells, we used a genome-editing approach. By Agrobacterium-mediated genetic transformation of immature embryos of cv. Avans, we obtained 14 transgenic plants with genetic constructs for site-directed mutagenesis of the k1C5 and g KAF1 genes encoding 22 k Da a-and 28 kDa γ-kafirins, respectively. Sequencing of 5 regenerants obtained by using k1C5-addressing vector revealed two plants with mutations. T_1 progeny of these mutants had higher in vitro digestibility of endosperm proteins(86%–92%), in comparison with the donor Avans(63%–67%). The kernels of these plants had a thick vitreous endosperm. A mutant with increased in vitro protein digestibility and vitreous endosperm, carrying a mutation in the target sequence, was also obtained by use of the gKAF1-addressing vector. Thus, using genome editing technology, we have obtained mutants with improved kafirin digestibility that can be used in sorghum breeding.展开更多
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 immature embryo explants.展开更多
基金supported by the Russian Foundation for Basic Research (grant#19-016-00117)。
文摘Sorghum(Sorghum bicolor(L.) Moench) is a major world crop that is a reliable source of fodder and food grain in arid regions. However, unlike other cereals, sorghum grain has low nutritional value, owing mainly to the resistance of its storage proteins(kafirins) to protease digestion. Changing the composition of kafirins or their primary structure may address this problem. To induce mutations in kafirin-encoding genes that were expected to disturb their accumulation in endosperm cells, we used a genome-editing approach. By Agrobacterium-mediated genetic transformation of immature embryos of cv. Avans, we obtained 14 transgenic plants with genetic constructs for site-directed mutagenesis of the k1C5 and g KAF1 genes encoding 22 k Da a-and 28 kDa γ-kafirins, respectively. Sequencing of 5 regenerants obtained by using k1C5-addressing vector revealed two plants with mutations. T_1 progeny of these mutants had higher in vitro digestibility of endosperm proteins(86%–92%), in comparison with the donor Avans(63%–67%). The kernels of these plants had a thick vitreous endosperm. A mutant with increased in vitro protein digestibility and vitreous endosperm, carrying a mutation in the target sequence, was also obtained by use of the gKAF1-addressing vector. Thus, using genome editing technology, we have obtained mutants with improved kafirin digestibility that can be used in sorghum breeding.
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 immature embryo explants.
