Liu Xingdan, Ding Qi, Wang Wenshu, Pan Yanling, Tan Chao, Qiu Yingbo, Chen Ya, Li Hongjing, Li Yinlong, Ye Naizhong, Xu Nian, Wu Xiao, Ye Rongjian, Liu Jianfeng, Ma Chonglie
College of Agronomy, Hunan Agricultural University, Changsha, 410128, China.
Life Science and Technology Center, China National Seed Group Co., LTD, Wuhan, 430206, Hubei, China.
Rice (N Y). 2022 Jan 4;15(1):1. doi: 10.1186/s12284-021-00548-y.
The rice Waxy (Wx) gene plays a major role in seed amylose synthesis and consequently controls grain amylose content. Wx gene expression is highly regulated at the post-transcriptional level. In particular, the GT/TT polymorphism at the 5'splicing site of its 1st intron greatly affects this intron's splicing efficiency and defines two predominant Wx alleles, Wx and Wx. Wx rice often harbours intermediate to high amylose contents, whereas Wx rice exhibits low to intermediate amylose contents. By deleting the Wx 1st intron using CRISPR/Cas9 technology, we generate a completely novel Wx allele and further investigate how intron removal affects Wx gene expression and rice grain amylose content.
CRISPR/Cas9-mediated targeted deletion of the Wx 1st intron was performed on 4 rice inbred lines: KY131 (Wx), X32 (Wx), X35 (Wx) and X55 (Wx). Deletion of the 1st intron occurred in 8.6-11.8% of the primary transformants of these 4 inbred lines. Compared to wild-type plants, amylose content was significantly increased from 13.0% to approximately 24.0% in KY131 and X32 mutant lines, which both carried the Wx allele. However, no significant difference in amylose content was observed between wild-type plants and X35 and X55 mutant lines, which carried the Wx and Wx alleles, respectively. Wx gene expression analysis of wild-type plants and mutants yielded results that were highly consistent with amylose content results. KY131 and X32 mutants accumulated increased levels of steady mRNA transcripts compared with wild-type plants, whereas steady mRNA levels were not altered in X35 and X55 mutants compared with wild-type plants. Grain quality, including appearance quality and eating and cooking quality, which are tightly associated with amylose content, was also assessed in wild-type and mutant plants, and data were presented and analysed.
This study presents a novel and rapid strategy to increase amylose content in inbred rice carrying a Wx allele. Our data strongly suggest that the 1st intron of the Wx gene regulates Wx gene expression mainly at the post-transcriptional level in rice. This finding is in contrast to a previous hypothesis suggesting that it influences Wx gene transcription. In addition, removal of the first intron generates a completely novel Wx allele. Further studies on this new Wx allele will provide invaluable insights into the regulation of Wx gene expression, which will help researchers engineer new Wx alleles to facilitate the breeding of rice cultivars with better eating and cooking quality.
水稻蜡质（Wx）基因在种子直链淀粉合成中起主要作用，从而控制籽粒直链淀粉含量。Wx基因的表达在转录后水平受到高度调控。特别是，其第1内含子5'剪接位点的GT/TT多态性极大地影响该内含子的剪接效率，并定义了两个主要的Wx等位基因，Wx和Wx。Wx水稻通常具有中等至高直链淀粉含量，而Wx水稻表现出低至中等直链淀粉含量。通过使用CRISPR/Cas9技术删除Wx第1内含子，我们产生了一个全新的Wx等位基因，并进一步研究内含子去除如何影响Wx基因表达和水稻籽粒直链淀粉含量。
在4个水稻自交系上进行了CRISPR/Cas9介导的Wx第1内含子靶向缺失：KY131（Wx）、X32（Wx）、X35（Wx）和X55（Wx）。这4个自交系的初级转化体中有8.6%-11.8%发生了第1内含子的缺失。与野生型植株相比，携带Wx等位基因的KY131和X32突变系的直链淀粉含量从13.0%显著增加到约24.0%。然而，分别携带Wx和Wx等位基因的野生型植株与X35和X55突变系之间的直链淀粉含量没有显著差异。野生型植株和突变体的Wx基因表达分析结果与直链淀粉含量结果高度一致。与野生型植株相比，KY131和X32突变体积累了更高水平的稳定mRNA转录本，而与野生型植株相比，X35和X55突变体的稳定mRNA水平没有改变。还对野生型和突变体植株的籽粒品质进行了评估，包括外观品质以及与直链淀粉含量密切相关的食味和蒸煮品质，并展示和分析了数据。
本研究提出了一种新颖且快速的策略来提高携带Wx等位基因的自交水稻的直链淀粉含量。我们的数据强烈表明，Wx基因的第1内含子主要在转录后水平调控水稻中的Wx基因表达。这一发现与之前认为它影响Wx基因转录的假设相反。此外，去除第一个内含子产生了一个全新的Wx等位基因。对这个新的Wx等位基因的进一步研究将为Wx基因表达的调控提供宝贵的见解，这将有助于研究人员设计新的Wx等位基因，以促进具有更好食味和蒸煮品质的水稻品种的培育。
