College of Forestry, Shenyang Agricultural University, Shenyang, China.
Key Laboratory of Forest Tree Genetics, Breeding and Cultivation of Liaoning Province, Shenyang, China.
Theor Appl Genet. 2022 Jun;135(6):1855-1865. doi: 10.1007/s00122-022-04079-y. Epub 2022 Apr 2.
A CRISPR/Cas9-based knockout assay verified that BoDFR1 drives anthocyanin accumulation in ornamental kale and that BoDFR2, an ortholog of BoDFR1, is redundant. Anthocyanins are widely distributed in nature and give plants their brilliant colors. Leaf color is an important trait for ornamental kale. In this study, we measured anthocyanin contents and performed transcriptome deep sequencing (RNA-seq) of leaves from pink and green ornamental kale. We observed substantial differences in the expression levels of the two DIHYDROFLAVONOL 4-REDUCTASE-encoding genes BoDFR1 (Bo9g058630) and its ortholog BoDFR2 (Bo2g116380) between green-leaved and pink-leaved kale by RNA-seq and RT-qPCR. We cloned and sequenced BoDFR1 and BoDFR2 from both types of kale. We identified a 1-bp insertion in BoDFR1 and a 2-bp insertion in BoDFR2 in green-leaved kale compared to the sequences obtained from pink-leaved kale, both mapping to the second exon of their corresponding gene and leading to premature termination of translation. To confirm the genetic basis of the absence of anthocyanins in green kale, we used CRISPR/Cas9 genome editing to separately knock out BoDFR1 or BoDFR2 in the pink-leaved ornamental kale inbred line P23. We detected very low accumulation of anthocyanins in the resulting mutants Bodfr1-1 and Bodfr1-2, while Bodfr2-1 and Bodfr2-2 had anthocyanin levels comparable to those of the wild-type. We conclude that the insertion in BoDFR1, rather than that in BoDFR2, underlies the lack of anthocyanins in green-leaved ornamental kale. This work provides insight into the function of DFR and will contribute to germplasm improvement of ornamental plants.
基于 CRISPR/Cas9 的敲除实验验证了 BoDFR1 驱动羽衣甘蓝中的花色素苷积累，而 BoDFR2（BoDFR1 的同源基因）是冗余的。花色素苷广泛存在于自然界中，赋予植物鲜艳的颜色。叶片颜色是羽衣甘蓝观赏品种的一个重要特征。在这项研究中，我们测量了粉色和绿色羽衣甘蓝叶片中的花色素苷含量，并进行了转录组深度测序（RNA-seq）。我们通过 RNA-seq 和 RT-qPCR 观察到，在绿叶和粉绿叶羽衣甘蓝中，两个二氢黄酮醇 4-还原酶编码基因 BoDFR1（Bo9g058630）和其同源基因 BoDFR2（Bo2g116380）的表达水平存在显著差异。我们从两种羽衣甘蓝中克隆并测序了 BoDFR1 和 BoDFR2。我们发现，与从粉绿叶羽衣甘蓝中获得的序列相比，绿叶羽衣甘蓝中的 BoDFR1 有 1 个碱基插入，BoDFR2 有 2 个碱基插入，均位于相应基因的第二外显子，导致翻译提前终止。为了确认绿色羽衣甘蓝中缺乏花色素苷的遗传基础，我们使用 CRISPR/Cas9 基因组编辑分别敲除了粉色羽衣甘蓝自交系 P23 中的 BoDFR1 或 BoDFR2。我们在产生的突变体 Bodfr1-1 和 Bodfr1-2 中检测到花色素苷的积累非常低，而 Bodfr2-1 和 Bodfr2-2 的花色素苷水平与野生型相当。我们得出结论，导致绿叶羽衣甘蓝缺乏花色素苷的原因是 BoDFR1 中的插入，而不是 BoDFR2 中的插入。这项工作为 DFR 的功能提供了新的认识，并将为观赏植物的种质改良做出贡献。
