Zhao Pei, Liu Yuxia, Deng Zhuyun, Liu Lingtong, Yu Tengwei, Ge Gege, Chen Bingtang, Wang Tai
Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China; College of Life Science, University of Chinese Academy of Sciences, Beijing, China; China National Botanical Garden, Beijing, China.
Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China; College of Life Science, University of Chinese Academy of Sciences, Beijing, China.
J Plant Physiol. 2024 Dec;303:154384. doi: 10.1016/j.jplph.2024.154384. Epub 2024 Nov 21.
Granule-bound starch synthase I (GBSSI) encoding gene Waxy (Wx), which largely regulates the amylose content of rice grains, is a master module determining rice eating and cooking quality (ECQ). Fine-tuning amylose level of grains is an ideal strategy to improve rice quality. Through fine editing of Wx promoter and 5'UTR by CRISPR/Cas9 system, we created 14 types of novel Wx allelic variations, of which MT7 and MT13 were able to alter Wx expression and amylose content of grains. MT7 showed fragment deletion and base insertions in CAAT-boxes, hardly detectable expression levels of GBSSI mRNA and protein, and generated 5.87% amylose in grains. MT13 had fragment deletions in the A-box and the TATA-box, low expression levels of GBSSI mRNA and protein, and generated 9.61% amylose in grains. Besides of the amylose content, MT7 and MT13 significantly reduced protein content and increased lipid content of grains compared with Wx. A comparison of MT7, MT13 and other allelic lines demonstrated the importance of base insertion around the second CAAT-box and 31bp-deletion following the second TATA-box in modulating Wx expression. Thus, our study generated two novel Wx allelic variations which significantly alter Wx expression and amylose content of rice grains, providing not only new germplasms for soft rice breeding, but also insights into candidate cis elements of Wx.
颗粒结合型淀粉合成酶I（GBSSI）编码基因蜡质基因（Wx）在很大程度上调控着水稻籽粒的直链淀粉含量，是决定稻米食用和蒸煮品质（ECQ）的关键模块。微调籽粒直链淀粉水平是改善稻米品质的理想策略。通过CRISPR/Cas9系统对Wx启动子和5'非翻译区进行精准编辑，我们创造了14种新型Wx等位基因变异，其中MT7和MT13能够改变Wx的表达以及籽粒的直链淀粉含量。MT7在CAAT框中出现片段缺失和碱基插入，GBSSI mRNA和蛋白的表达水平几乎检测不到，籽粒中直链淀粉含量为5.87%。MT13在A框和TATA框中有片段缺失，GBSSI mRNA和蛋白表达水平较低，籽粒中直链淀粉含量为9.61%。与Wx相比，除直链淀粉含量外，MT7和MT13显著降低了籽粒的蛋白质含量并提高了脂质含量。MT7、MT13与其他等位基因系的比较表明，第二个CAAT框周围的碱基插入以及第二个TATA框后的31bp缺失在调节Wx表达方面的重要性。因此，我们的研究产生了两种新型Wx等位基因变异，它们显著改变了水稻籽粒的Wx表达和直链淀粉含量，不仅为软米育种提供了新的种质资源，也为Wx的候选顺式元件提供了见解。
