Takeuchi Ami, Ohnuma Mariko, Teramura Hiroshi, Asano Kenji, Noda Takahiro, Kusano Hiroaki, Tamura Koji, Shimada Hiroaki
Department of Biological Science and Technology, Tokyo University of Science, Katsushika, Tokyo 125-8585, Japan.
Division of Northern Field Crop Research, Field Crop Breeding Group, NARO, 9-4 Shinsei-minami, Memuro, Kasai, Hokkaido 082-0081, Japan.
Plant Biotechnol (Tokyo). 2021 Sep 25;38(3):345-353. doi: 10.5511/plantbiotechnology.21.0727a.
The potato tuber starch trait is changed depending on the composition of amylose and amylopectin. The amount of amylopectin is determined by the activity of the starch branching enzymes SBE1, SBE2, and SBE3 in potato. SBE3, a homolog of rice BEI, is a major gene that is abundant in tubers. In this study, we created mutants of the potato gene using CRISPR/Cas9 attached to the translation enhancer dMac3. Potato has a tetraploid genome, and a four-allele mutant of the gene is desired. Mutations in the gene were found in 89 of 126 transformants of potato plants. Among these mutants, 10 lines contained four mutant SBE3 genes, indicating that 8% efficiency of target mutagenesis was achieved. These mutants grew normally, similar to the wild-type plant, and yielded sufficient amounts of tubers. The potato starch in these tubers was similar to that of the rice BEI mutant. Western blot analysis revealed the defective production of SBE3 in the mutant tubers, suggesting that these transformants were loss-of-function mutants of .
马铃薯块茎淀粉特性会根据直链淀粉和支链淀粉的组成而发生变化。支链淀粉的含量由马铃薯中淀粉分支酶SBE1、SBE2和SBE3的活性决定。SBE3是水稻BEI的同源物，是在块茎中大量存在的一个主要基因。在本研究中，我们使用与翻译增强子dMac3相连的CRISPR/Cas9技术创建了马铃薯基因的突变体。马铃薯具有四倍体基因组，需要获得该基因的四等位基因突变体。在126株马铃薯转化植株中，有89株被发现该基因发生了突变。在这些突变体中，有10个株系含有4个突变的SBE3基因，这表明实现了8%的靶向诱变效率。这些突变体生长正常，与野生型植株相似，并且能产生足够数量的块茎。这些块茎中的马铃薯淀粉与水稻BEI突变体的淀粉相似。蛋白质免疫印迹分析显示突变块茎中SBE3的产生存在缺陷，这表明这些转化体是该基因的功能缺失突变体。
