Plant Biotechnology Journal:农科院作科所谢传晓研究组发表玉米基因组
2017年4月5日，《plant Biotechnology Journal》期刊在线发表了中国农业科学院作物科学研究所玉米分子育种技术和应用创新团队玉米基因组编辑育种创新研究组谢传晓研究员题为“RNA-guided Cas9 as an in vivo desired-target mutator in maize”的研究论文，报道了该小组利用基因编辑技术提高作物育种效率的研究实例及其具体实现途径。硕士生李楚曦和刘昌林助理研究员为论文的共同第一作者，谢传晓研究员为论文通讯作者。
传统育种过程中，目标基因在受体品种中的聚合与导入依赖减数分裂过程中的遗传重组与交换，不可回避会面临连锁累赘(linkage drag)问题，显著影响育种效率，需要多个遗传世代从构建较大的遗传群体中筛选与鉴定目标基因两侧发生供体与受体DNA双交换的小概率遗传事件，以实现目标导入聚合且受体遗传背景回复。多种作物回交育种实践都表明，采用多个遗传世代回交与大群体筛选回交育种策略仍难以克服连锁累赘问题，育种过程耗时长，工作量大，效率低。
该研究组采用基于CRISPR原理的RNA指导的Cas9核酸酶玉米基因编辑技术对玉米LG1(LIGULELESS1)基因第1外显子序列进行定向突变，产生基因敲除突变，定向突变率达到51.5%至91.2%。研究人员把携带定向编辑LG1工具的玉米植株与一系列受体杂交，通过植株活体基因编辑工具定向编辑受体目标基因，实现了达到11.79%至28.71%活体目标基因编辑定点突变活性，且可以稳定遗传。目标基因突变后，植株突变表型明显，叶片夹角表型减小至对照的50%。田间试验还表明该突变紧凑株型表型具备通过增密从而实现增产的潜力。该研究证实了植株携带的基因编辑工具在配子体和孢子体水平存在很高的活性，可以应用于玉米育种。针对玉米遗传转化困难且严重依赖基因型的特点，该研究实现了有效利用基因编辑进行高效定向突变育种，为推动基因组编辑技术育种应用提供了成功的范例。
原文链接：
RNA-guided Cas9 as an in vivo desired-target mutator in maize
原文摘要：
The RNA-guided Cas9 system is a versatile tool for genome editing. Here, we established a RNA-guided endonuclease (RGEN) system as an in vivo desired-target mutator (DTM) in maize to reduce the linkage drag during breeding procedure, using the LIGULELESS1 (LG1) locus as a proof-of-concept. Our system showed 51.5% to 91.2% mutation frequency in T0 transgenic plants. We then crossed the T1 plants stably expressing DTM with six diverse recipient maize lines and found that 11.79% to 28.71% of the plants tested were mutants induced by the DTM effect. Analysis of successive F2 plants indicated that the mutations induced by the DTM effect were largely heritable. Moreover, DTM-GENErated hybrids had significantly smaller leaf angles that were reduced more than 50% when compared with that of the wild type. Planting experiments showed that DTM generated maize plants can be grown with significantly higher density and hence greater yield potential. Our work demonstrate that stably expressed RGEN could be implemented as an in vivo DTM to rapidly generate and spread desired mutations in maize through hybridization and subsequent backcrossing, and hence bypassing the linkage drag effect in convention introgression methodology. This proof-of-concept experiment can be a potentially much more efficient breeding strategy in crops employing the RNA-guided Cas9 genome editing.
作者：谢传晓
