Tuskegee University, Tuskegee, AL, 36088, USA.
Shandong Peanut Research Institute, Qingdao, 266100, China.
BMC Biotechnol. 2019 Apr 29;19(1):24. doi: 10.1186/s12896-019-0516-8.
Increasing the content of oleic acid in peanut seeds is one of the major goals in peanut breeding due to consumer and industry benefits, such as anti-oxidation and long shelf-life. Homeologous ahFAD2A and ahFAD2B genes encode fatty acid desaturases, which are the key enzymes for converting oleic acid to linoleic acid that oxidizes readily. To date, all high oleic acid peanut varieties result from natural mutations occurred in both genes. A method to induce mutations in the genes of other elite cultivars could speed introgression of this valuable trait. The gene-editing approach utilizing CRISPR/Cas9 technology was employed to induce de novo mutations in the ahFAD2 genes using peanut protoplasts and hairy root cultures as models.
The hot spot of natural mutation in these genes was selected as the target region. Appropriate sgRNAs were designed and cloned into a CRISPR/Cas9 expression plasmid. As a result of CRISPR/Cas9 activity, three mutations were identified - G448A in ahFAD2A, and 441_442insA and G451T in ahFAD2B. The G448A and 441_442insA mutations are the same as those seen in existing high oleate varieties and the G451T is new mutation. Because natural mutations appear more often in the ahFAD2A gene than in the ahFAD2B gene in subspecies A. hypogaea var. hypogaea, the mutations induced in ahFAD2B by gene editing may be useful in developing high oleate lines with many genetic backgrounds after validation of oleic acid content in the transformed lines. The appearance of the G448A mutation in ahFAD2A is a further benefit for high oleic acid oil content.
Overall, these results showed that mutations were, for the first time, induced by CRISPR-based gene editing approach in peanut. This research demonstrated the potential application of gene editing for mutagenesis in peanut and suggested that CRISPR/Cas9 technology may be useful in the peanut breeding programs.
提高花生种子中油酸的含量是花生育种的主要目标之一，因为这对消费者和行业都有好处，例如抗氧化和延长保质期。同源 ahFAD2A 和 ahFAD2B 基因编码脂肪酸去饱和酶，是将油酸转化为容易氧化的亚油酸的关键酶。迄今为止，所有高油酸花生品种都是由这两个基因中的自然突变产生的。在其他优良品种的基因中诱导突变的方法可以加速这一有价值性状的导入。该研究利用 CRISPR/Cas9 技术的基因编辑方法，在花生原生质体和毛状根培养物作为模型中，在 ahFAD2 基因中诱导从头突变。
选择这些基因中的自然突变热点作为靶区域。设计并将适当的 sgRNA 克隆到 CRISPR/Cas9 表达质粒中。由于 CRISPR/Cas9 的活性，鉴定出了三个突变 - ahFAD2A 中的 G448A，以及 ahFAD2B 中的 441_442insA 和 G451T。G448A 和 441_442insA 突变与现有高油酸品种中的突变相同，而 G451T 是新的突变。由于在 A. hypogaea var. hypogaea 亚种中，ahFAD2A 基因中的自然突变比 ahFAD2B 基因更常见，因此在 ahFAD2B 基因中诱导的突变在验证转化系中油酸含量后，可能有助于开发具有多种遗传背景的高油酸系。ahFAD2A 中 G448A 突变的出现是高油酸油含量的进一步好处。
总之，这些结果表明，首次通过基于 CRISPR 的基因编辑方法在花生中诱导了突变。该研究证明了基因编辑在花生诱变中的潜在应用，并表明 CRISPR/Cas9 技术可能在花生育种计划中有用。
