Virdi Kamaldeep S, Spencer Madison, Stec Adrian O, Xiong Yer, Merry Ryan, Muehlbauer Gary J, Stupar Robert M
Department of Agronomy & Plant Genetics, University of Minnesota, Saint Paul, MN, United States.
Front Plant Sci. 2020 Jul 8;11:1005. doi: 10.3389/fpls.2020.01005. eCollection 2020.
The β-ketoacyl-[acyl carrier protein] synthase 1 () gene has been shown in model plant systems to be critical for the conversion of sucrose to oil. A previous study characterized the morphological and seed composition phenotypes associated with a reciprocal chromosomal translocation that disrupted one of the genes in soybean. The principle findings of this work included a wrinkled seed phenotype, an increase in seed sucrose, a decrease in seed oil, and a low frequency of transmission of the translocation. However, it remained unclear which, if any, of these phenotypes were directly caused by the loss of gene function, as opposed to the chromosomal translocation or other associated factors. In this study, CRISPR/Cas9 mutagenesis was used to generate multiple knockout alleles for this gene, and also one in-frame allele. These soybean plants were evaluated for morphology, seed composition traits, and genetic transmission. Our results indicate that the CRISPR/Cas9 mutants exhibited the same phenotypes as the chromosomal translocation mutant, validating that the observed phenotypes are caused by the loss of gene function. Furthermore, the plants harboring homozygous in-frame mutations exhibited similar phenotypes compared to the plants harboring homozygous knockout mutations. This result indicates that the amino acids lost in the in-frame mutant are essential for proper gene function. In-frame edits for this gene may need to target less essential and/or evolutionarily conserved domains in order to generate novel seed composition phenotypes.
β-酮脂酰-[酰基载体蛋白]合酶1（）基因在模式植物系统中已被证明对蔗糖向油的转化至关重要。先前的一项研究对与一个相互染色体易位相关的形态和种子成分表型进行了表征，该易位破坏了大豆中的一个基因。这项工作的主要发现包括皱粒种子表型、种子蔗糖增加、种子油减少以及易位的低传递频率。然而，尚不清楚这些表型中哪些（如果有的话）是由基因功能丧失直接导致的，而不是由染色体易位或其他相关因素导致的。在本研究中，利用CRISPR/Cas9诱变技术为该基因产生了多个敲除等位基因，以及一个框内等位基因。对这些大豆植株进行了形态、种子成分性状和遗传传递评估。我们的结果表明，CRISPR/Cas9突变体表现出与染色体易位突变体相同的表型，证实了观察到的表型是由基因功能丧失引起的。此外，与携带纯合敲除突变的植株相比，携带纯合框内突变的植株表现出相似的表型。这一结果表明，框内突变体中缺失的氨基酸对正常基因功能至关重要。对该基因进行框内编辑可能需要针对不太重要和/或进化上保守的结构域，以产生新的种子成分表型。
