National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China.
Hubei Key Laboratory of Food Crop Germplasm and Genetic Improvement, Food Crops Institute, Hubei Academy of Agricultural Sciences, Wuhan 430064, China.
Int J Mol Sci. 2022 Nov 16;23(22):14165. doi: 10.3390/ijms232214165.
Type-B response regulator proteins in rice contain a conserved receiver domain, followed by a GARP DNA binding domain and a longer C-terminus. Some type-B response regulators such as RR21, RR22 and RR23 are involved in the development of rice leaf, root, flower and trichome. In this study, to evaluate the application potential of type-B response regulators in rice genetic improvement, thirteen type-B response regulator genes in rice were respectively knocked out by using CRISPR/Cas9 genome editing technology. Two guide RNAs (gRNAs) were simultaneously expressed on a knockout vector to mutate one gene. T transformed plants were used to screen the plants with deletion of large DNA fragments through PCR with specific primers. The mutants of CRISPR/Cas9 gene editing were detected by specific primer in the T generation, and homozygous mutants without were screened, whose target regions were confirmed by sequencing. Mutant materials of 12 were obtained, except for . Preliminary phenotypic observation revealed variations of various important traits in different mutant materials, including plant height, tiller number, tillering angle, heading date, panicle length and yield. The mutant in the T generation was then further examined. As a result, the heading date of the mutant was delayed by about 18 d, while the yield was increased by about 30%, and the chalkiness was significantly reduced compared with those of the wild-type under field high temperature stress. These results indicated that has great application value in rice breeding. Our findings suggest that it is feasible to perform genetic improvement of rice by editing the type-B response regulators.
水稻中的 B 型反应调节蛋白含有一个保守的受体结构域，其后是 GARP DNA 结合结构域和更长的 C 末端。一些 B 型反应调节蛋白，如 RR21、RR22 和 RR23，参与了水稻叶片、根系、花和毛状体的发育。在这项研究中，为了评估 B 型反应调节蛋白在水稻遗传改良中的应用潜力，我们利用 CRISPR/Cas9 基因组编辑技术分别敲除了水稻中的 13 个 B 型反应调节基因。一个敲除载体上同时表达两个向导 RNA（gRNA），以突变一个基因。使用 T 转化植株通过使用特定引物的 PCR 筛选具有大片段 DNA 缺失的植株。在 T 代中通过特异性引物检测 CRISPR/Cas9 基因编辑的突变体，并筛选出没有的纯合突变体，其靶区域通过测序确认。除了以外，获得了 12 个突变体材料。初步表型观察显示，不同突变体材料的各种重要性状都发生了变化，包括株高、分蘖数、分蘖角度、抽穗期、穗长和产量。然后进一步研究了 T 代中的突变体。结果表明，突变体的抽穗期延迟了约 18 天，而产量增加了约 30%，与野生型相比，在田间高温胁迫下，垩白度显著降低。这些结果表明在水稻育种中具有很大的应用价值。我们的研究结果表明，通过编辑 B 型反应调节蛋白可以实现水稻的遗传改良。
