Creation of Early Flowering Germplasm of Soybean by CRISPR/Cas9 Technology
Creation of Early Flowering Germplasm of Soybean by CRISPR/Cas9 Technology
复制标题
DOI：
10.3389/fpls.2019.01446
复制
发表时间：
2019-11-22
影响因子：
5.6
通讯作者：
Qiu, Li-Juan
中科院分区：
文献类型：
--
作者：
Han, Jianan;Guo, Bingfu;Qiu, Li-Juan
大豆是一种重要的经济作物，也是典型的短日照作物，对光周期敏感，地理适应范围窄，限制了转基因材料的产生，降低了新品种的选育效率。此外，大豆的遗传转化效率低于许多其他作物，可利用的受体基因类型有限。本研究利用农杆菌介导法将CRISPR/Cas9表达载体导入大豆品种JACK，获得了控制大豆开花的E1基因的靶向突变体。我们获得了两种新的突变类型，分别是E1编码区11bp和40bp的缺失，移码突变导致提前翻译终止密码子和截短的E1蛋白，导致在长日照条件下明显的提前开花。此外，通过预测和分析E1靶的潜在脱靶部位，未观察到脱靶效应。两个新突变体的E1基因表达显著降低，表明截短的E1蛋白抑制了GmFT2a/5a的表达，增加了GmFT2a/5a的基因表达，导致了明显的早花。还获得了不含T-DNA元件的纯合反式清洁突变体，在长日照条件下表现出较早的开花。我们创造的光不敏感大豆转化受体为选育适应高纬度地区的优良转基因受体奠定了基础。
Soybean is an important economic crop and a typical short-day crop, sensitive to photoperiod, and has narrow geographical adaptative region, which limit the creation of transgenic materials and reduce the breeding efficiency of new varieties. In addition, the genetic transformation efficiency of soybean is lower than that of many other crops, and the available receptor genotypes are limited. In this study, Agrobacterium-mediated transformation were used to introduce the CRISPR/Cas9 expression vector into soybean cultivar Jack and generated targeted mutants of E1 gene controlling soybean flowering. We obtained two novel types of mutations, 11 bp and 40 bp deletion at E1 coding region, respectively, and frameshift mutations produced premature translation termination codons and truncated E1 proteins, causing obvious early flowering under long day condition. In addition, no off-target effects were observed by predicting and analyzing the potential off-target sites of E1 targets. Significant decreased E1 gene expression of two novel mutants showed that the truncated E1 protein disinhibited GmFT2a/5a and increasing GmFT2a/5a gene expressions resulted obvious early flowering. Homozygous trans-clean mutants without T-DNA elements were also obtained and showed early flowering under long day condition. The photo-insensitive soybean transformation receptor we created laid a foundation for breeding excellent transgenic receptors suitable for high latitudes.