Allele exchange at the EPSPS locus confers glyphosate tolerance in cassava.
Allele exchange at the EPSPS locus confers glyphosate tolerance in cassava.
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DOI：
10.1111/pbi.12868
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发表时间：
2018-07
影响因子：
13.8
通讯作者：
Taylor NJ
中科院分区：
文献类型：
--
作者：
Hummel AW;Chauhan RD;Cermak T;Mutka AM;Vijayaraghavan A;Boyher A;Starker CG;Bart R;Voytas DF;Taylor NJ
有效的杂草防除可以保护木薯贮藏根部的产量。农民可以从使用耐受品种的除草剂中受益。我们应用传统的转基因和基因编辑在木薯中产生了强大的草甘膦耐受性。通过比较转化的5-烯醇丙酮基莽草酸-3-磷酸合成酶(EPSPS)基因的启动子和不同氨基酸对的替代，我们发现，在体外选择和整个木薯植株中，获得草甘膦耐性需要强大的结构性表达。利用同源重组(HR)和非同源末端连接(NHEJ)DNA修复途径，我们精确地将表现最好的等位基因导入木薯基因组，同时在内源EPSPS基因座上创建启动子交换和双氨基酸替换。EPSPS编辑的初代植物表型正常，对高剂量草甘膦耐受，有些没有可检测到的T-DNA整合。我们的方法展示了一种在农作物中创造草甘膦耐性的编辑策略，并展示了进一步改良木薯的基因编辑的潜力。
Effective weed control can protect yields of cassava (Manihot esculenta) storage roots. Farmers could benefit from using herbicide with a tolerant cultivar. We applied traditional transgenesis and gene editing to generate robust glyphosate tolerance in cassava. By comparing promoters regulating expression of transformed 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) genes with various paired amino acid substitutions, we found that strong constitutive expression is required to achieve glyphosate tolerance during in vitro selection and in whole cassava plants. Using strategies that exploit homologous recombination (HR) and nonhomologous end‐joining (NHEJ) DNA repair pathways, we precisely introduced the best‐performing allele into the cassava genome, simultaneously creating a promoter swap and dual amino acid substitutions at the endogenous EPSPS locus. Primary EPSPS‐edited plants were phenotypically normal, tolerant to high doses of glyphosate, with some free of detectable T‐DNA integrations. Our methods demonstrate an editing strategy for creating glyphosate tolerance in crop plants and demonstrate the potential of gene editing for further improvement of cassava.
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