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Bina.y Vector Construction for Site-Directed Muta.enesis of <i>Ka.irin</i>Genes in Sorghum

a.erica. Journa. of Pla.t Sciences 2021年第8期12卷 1276-1287页

作者： grigoriy a. gerashchenkov Lev a. Elkonin Kirill G. gerashchenkov Na.a.ia.a. Rozhnova.Stefa. Hiekel Jochen Kumlehn a.exey V. Chemeris Institute of Biochemistry a.d Genetics Subdivision of the Ufa Federal Research Centre of the Russian Academy of Sciences Ufa Russia Federa. Centre of a.riculture Resea.ch of South-Ea.t Region Saratov Russia Ka.a. Federa. University Institute of Fundamental Medicine and Biology Kazan Russia Leibniz Institute of Pla.t Genetics a.d Crop Pla.t Resea.ch (IPK) Gatersleben Germany

Sorghum (Sorghum bicolor (L.) Moench) is one of the world’s lea.ing cerea. crops in a.ricultura. production, which ha. a.specia. importa.ce in the a.id regions. However, unlike other cerea.s, sorghum gra.n ha. a.lower nutritiona. va.ue, which is ca.sed, inter a.ia. by the resista.ce of its seed stora.e proteins (ka.irins) to protea.e digestion. One of the effective a.proa.hes to improve the nutritiona. va.ue of sorghum gra.n is to obta.n muta.ts with pa.tia.ly or completely suppressed synthesis or a.tered a.ino a.id composition of ka.irins. The employment of genome editing ma. a.low to solve this problem by introducing muta.ions into the nucleotide sequences of the α- a.d γ-ka.irin genes. In this study, genomic ta.get motifs (23 bp sequences) were selected for the introduction of muta.ions into the α- a.d γ-Ka.IRIN genes of sorghum. The design of the gRNAs was conducted using the online tools CRISPROR a.d CHOPCHOP. a.na.e="_Hlk55317737">a.Two most suita.le ta.gets were chosen for α-Ka.IRIN (k1C5) a.d two for γ-Ka.IRIN (gKa.1) genes. The insertion of respective sequences in the generic vector pSH121 wa. performed a. the Bsa. (Eco31I) sites. Va.ida.ion of the cloning procedure wa. performed by DNa.sequencing. Subcloning of the resulting constructs wa. performed using the SfiI restriction sites into the compa.ible bina.y vector B479p7oUZm-LH. The correct a.sembly of bina.y vectors wa. confirmed by restriction a.a.ysis using the MluI a.d SfiI clea.a.e sites. The four vectors crea.ed (1Ca.> - 4C) were transferred by electroporation into the Agrobacterium tumefaciens stra.n a.L0. Currently, this vector series is used for sta.le

关键词： Sorghum bicolor (L.) Moench CRISPR/Ca. Genome Editing α-Ka.irin γ-Ka.irin Genetic Engineering Gra.n Qua.ity

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Development of sorghum muta.ts with improved in vitro protein digestibility by CRISPR/Ca.9 editing of ka.irin genes

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The Crop Journa. 2023年第5期11卷 1411-1418页

作者： Lev a.Elkonin grigoriy a.gerashchenkov Na.a.ie V.Borisenko Odyssey a.Kenzhegulov Sa.le Kh.Sa.senova.Na.a.ya.a.Rozhnova.Va.ery M.Pa.in Federa. Centre of a.riculture Resea.ch of South-Ea.t Region Saratov 410010Russia Institute of Biochemistry a.d Genetics-Subdivision of the Ufa.Federa. Resea.ch Centre of the Russia. a.a.emy of Sciences Ufa 450054Russia

Sorghum(Sorghum bicolor(L.) Moench) is a.ma.or world crop tha. is a.relia.le source of fodder a.d food gra.n in a.id regions. However, unlike other cerea.s, sorghum gra.n ha. low nutritiona. va.ue, owing ma.nly to the resista.ce of its stora.e proteins(ka.irins) to protea.e digestion. Cha.ging the composition of ka.irins or their prima.y structure ma. a.dress this problem. To induce muta.ions in ka.irin-encoding genes tha. were expected to disturb their a.cumula.ion in endosperm cells, we used a.genome-editing a.proa.h. By a.roba.terium-media.ed genetic tra.sforma.ion of imma.ure embryos of cv. a.a.s, we obta.ned 14 tra.sgenic pla.ts with genetic constructs for site-directed muta.enesis of the k1C5 a.d g Ka.1 genes encoding 22 k Da.a.a.d 28 kDa.γ-ka.irins, respectively. Sequencing of 5 regenera.ts obta.ned by using k1C5-a.dressing vector revea.ed two pla.ts with muta.ions. T_1 progeny of these muta.ts ha. higher in vitro digestibility of endosperm proteins(86%–92%), in compa.ison with the donor a.a.s(63%–67%). The kernels of these pla.ts ha. a.thick vitreous endosperm. a.muta.t with increa.ed in vitro protein digestibility a.d vitreous endosperm, ca.rying a.muta.ion in the ta.get sequence, wa. a.so obta.ned by use of the gKa.1-a.dressing vector. Thus, using genome editing technology, we ha.e obta.ned muta.ts with improved ka.irin digestibility tha. ca. be used in sorghum breeding.

关键词： Sorghum CRISPR/Ca. Ka.irins In vitro protein digestibility Vitreous endosperm

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