Kishi-Kaboshi Mitsuko, Abe Fumitaka, Kamiya Yoko, Kawaura Kanako, Hisano Hiroshi, Sato Kazuhiro
Institute of Crop Science, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki 305-8518, Japan.
Kihara Institute for Biological Research, Yokohama City University, Yokohama, Kanagawa 244-0813, Japan.
Plant Biotechnol (Tokyo). 2023 Sep 25;40(3):237-245. doi: 10.5511/plantbiotechnology.23.0717a.
Genome editing is a promising method for simultaneously mutagenizing homoeologs in the three subgenomes of wheat ( L.). However, the mutation rate via genome editing must be improved in order to analyze gene function and to quickly modify agronomic traits in wheat. Here, we examined the Cas9-induced mutation rates in wheat plants using two promoters for single guide RNA (sgRNA) expression and applying heat treatment during -mediated transformation. Using the promoter instead of the promoter from rice ( L.) to drive sgRNA expression greatly improved the Cas9-induced mutation rate. Moreover, a heat treatment of 30°C for 1 day during tissue culture increased the Cas9-induced mutation rate and the variety of mutations obtained compared to tissue culture at the normal temperature (25°C). The same heat treatment did not affect the regeneration rates of transgenic plants but tended to increase the number of transgene integration sites in each transgenic plant. These results lay the foundation for improving the Cas9-induced mutation rate in wheat to enhance research on gene function and crop improvement.
基因组编辑是一种很有前景的方法，可用于同时诱变小麦（L.）三个亚基因组中的同源基因。然而，为了分析基因功能并快速改良小麦的农艺性状，必须提高通过基因组编辑产生的突变率。在这里，我们使用两个用于单向导RNA（sgRNA）表达的启动子，并在介导转化过程中进行热处理，检测了Cas9诱导的小麦植株突变率。使用启动子而非水稻（L.）的启动子来驱动sgRNA表达，极大地提高了Cas9诱导的突变率。此外，与在常温（25°C）下进行组织培养相比，在组织培养期间进行30°C处理1天可提高Cas9诱导的突变率以及获得的突变种类。相同的热处理不影响转基因植株的再生率，但倾向于增加每个转基因植株中转基因整合位点的数量。这些结果为提高小麦中Cas9诱导的突变率奠定了基础，以加强对基因功能的研究和作物改良。
