Asa Hiroki, Kuwabara Chikako, Matsumoto Kenji, Shigeta Ryo, Yamamoto Takaaki, Masuda Yu, Yamada Tetsuya
Graduate School of Agriculture, Hokkaido University, Kita 9, Nishi 9, Kita-ku, Sapporo, Hokkaido, 060-8589, Japan.
Kanematsu Corporation, Marunouchi 2-7-2, Chiyoda-ku, Tokyo, 100-7017, Japan.
Plant Cell Rep. 2025 Jan 28;44(2):40. doi: 10.1007/s00299-025-03433-w.
We generated soybean mutants related to two ß-amyrin synthase genes using DNA-free site-directed mutagenesis system. Our results suggested that one of the genes is predominant in the soyasaponin biosynthesis. Soyasaponins, which are triterpenoid saponins contained in soybean [Glycine max (L.) Merril], are responsible for the astringent aftertaste of soyfood, and their complete elimination from soybean seeds is a key challenge in the development of cultivars with improved taste. While the loss of function in the ß-amyrin synthase genes (GmBAS1 and GmBAS2) has proven effective in reducing soyasaponin content in soybean seeds, the specific functional roles of these genes remain unclear. In this study, site-directed mutagenesis was performed on two GmBAS loci using a DNA-free clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system. A complex of sgRNA targeting sequences conserved in the two loci and Cas9 protein was introduced into the shoot apical meristems of soybean embryonic axes via bombardment. Cleaved amplified polymorphic sequences (CAPS) analysis conducted 1 month post-bombardment revealed that 138 seedlings out of 1,467 screened exhibited mutations at one or both GmBAS loci. CAPS and sequencing analysis in the subsequent generation identified a total of 16 plants with inheritable mutations ranging from one to ten nucleotides. High-performance liquid chromatography (HPLC) analysis showed that site-directed mutagenesis in the GmBAS1 locus resulted in the absence of soyasaponins in mature seeds, as well as in young roots, stems, and leaves. These findings demonstrate that GmBAS1 is the predominant ß-amyrin synthase gene in soybean plants. In addition, the DNA-free CRISPR/Cas9 system was shown to be highly efficient in inducing simultaneous mutagenesis at two target loci using a single gRNA.
我们使用无DNA定点诱变系统生成了与两个β-香树脂醇合酶基因相关的大豆突变体。我们的结果表明，其中一个基因在大豆皂素生物合成中起主要作用。大豆皂素是大豆[Glycine max (L.) Merril]中含有的三萜皂苷，是豆制品涩味的来源，从大豆种子中完全去除大豆皂素是培育口感改良品种的关键挑战。虽然β-香树脂醇合酶基因（GmBAS1和GmBAS2）功能丧失已被证明可有效降低大豆种子中的大豆皂素含量，但其具体功能作用仍不清楚。在本研究中，使用无DNA的成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关核酸内切酶9（Cas9）系统对两个GmBAS基因座进行了定点诱变。通过轰击将靶向两个基因座保守序列的sgRNA与Cas9蛋白的复合物导入大豆胚轴的茎尖分生组织。轰击后1个月进行的酶切扩增多态性序列（CAPS）分析表明，在筛选的1467株幼苗中，有138株在一个或两个GmBAS基因座发生了突变。后续世代的CAPS和测序分析共鉴定出16株具有可遗传突变的植株，突变范围为1至10个核苷酸。高效液相色谱（HPLC）分析表明，GmBAS1基因座的定点诱变导致成熟种子以及幼根、茎和叶中不存在大豆皂素。这些发现表明，GmBAS1是大豆植株中主要的β-香树脂醇合酶基因。此外，无DNA的CRISPR/Cas9系统被证明在使用单个gRNA同时诱导两个靶基因座的诱变方面效率很高。
