ABSTRACT

Genome editing tools have rapidly been adopted by plant scientists for crop improvement. Genome editing using a multiplex sgRNA-CRISPR/Cas9 genome editing system is a useful technique for crop improvement in monocot species. In this study, we utilized precise gene editing techniques to generate wheat 3’(2’), 5’-bisphosphate nucleotidase (TaSal1) mutants using a multiplex sgRNA-CRISPR/Cas9 genome editing system. Five active TaSal1 homologous genes were found in the genome of Giza168 in addition to another apparently inactive gene on chromosome 4A. Three gRNAs were designed and used to target exons 4, 5 and 7 of the five wheat TaSal1 genes. Among the 120 Giza168 transgenic plants, 41 lines exhibited mutations and produced heritable TaSal1 mutations in the M₁ progeny and 5 lines were full 5 gene knock-outs. These mutant plants exhibit a rolled-leaf phenotype in young leaves and bended stems, but there were no significant changes in the internode length and width, leaf morphology, and stem shape. Anatomical and scanning electron microscope studies of the young leaves of mutated TaSal1 lines showed closed stomata, increased stomata width and increase in the size of the bulliform cells. Sal1 mutant seedlings germinated and grew better on media containing polyethylene glycol than wildtype seedlings. Our results indicate that the application of the multiplex sgRNA-CRISPR/Cas9 genome editing is efficient tool for mutating more multiple TaSal1 loci in hexaploid wheat.

摘要

基因组编辑工具已迅速被植物科学家用于作物改良。使用多重 sgRNA-CRISPR/Cas9 基因组编辑系统进行基因组编辑是单子叶植物作物改良的有用技术。在这项研究中，我们利用精确的基因编辑技术，使用多重 sgRNA-CRISPR/Cas9 基因组编辑系统生成小麦 3'(2')、5'-二磷酸核苷酸酶 ( TaSal1 ) 突变体。在 Giza168 的基因组中发现了五个活跃的 TaSal1同源基因，此外还有一个在染色体 4A 上明显不活跃的基因。设计了三个 gRNA，并用于靶向五个小麦TaSal1基因的外显子 4、5 和 7 。在 120 株 Giza168 转基因植物中，41 株表现出突变并产生可遗传的 TaSal1M ₁后代和 5 个系中的突变是完整的 5 个基因敲除。这些突变植物在幼叶和弯曲茎中表现出卷叶表型，但在节间长度和宽度、叶片形态和茎形方面没有显着变化。对突变TaSal1株系幼叶的解剖和扫描电子显微镜研究显示气孔闭合，气孔宽度增加，泡状细胞大小增加。Sal1突变体幼苗在含有聚乙二醇的培养基上比野生型幼苗发芽和生长更好。我们的结果表明，多重 sgRNA-CRISPR/Cas9 基因组编辑的应用是在六倍体小麦中突变更多多个 TaSal1 基因座的有效工具。