Leng Fengjun, Zhou Guiwei, Shi Ruoyuan, Liu Chengyang, Lin Yirui, Yu Xinqiang, Zhang Yanhua, He Xiangxi, Liu Zhu, Sun Mingming, Bao Fang, Hu Yong, He Yikun
Key Laboratory of Plant Gene Resources and Biotechnology for Carbon Reduction and Environmental Improvement, Beijing Municipal Government, and College of Life Sciences, Capital Normal University, Beijing, 100048, China.
Laboratory for Micro-Sized Functional Materials, College of Elementary Education, Capital Normal University, Beijing, 100048, China.
Plant Cell Rep. 2024 Feb 10;43(3):63. doi: 10.1007/s00299-024-03143-9.
To establish a sterile culture system and protoplast regeneration system for Bryum argenteum, and to establish and apply CRISPR/Cas9 system in Bryum argenteum. Bryum argenteum is a fascinating, cosmopolitan, and versatile moss species that thrives in various disturbed environments. Because of its comprehensive tolerance to the desiccation, high UV and extreme temperatures, it is emerging as a model moss for studying the molecular mechanisms underlying plant responses to abiotic stresses. However, the lack of basic tools such as gene transformation and targeted genome modification has hindered the understanding of the molecular mechanisms underlying the survival of B. argenteum in different environments. Here, we reported the protonema of B. argenteum can survive up to 95.4% water loss. In addition, the genome size of B. argenteum is approximately 313 Mb by kmer analysis, which is smaller than the previously reported 700 Mb. We also developed a simple method for protonema induction and an efficient protoplast isolation and regeneration protocol for B. argenteum. Furthermore, we established a PEG-mediated protoplast transient transfection and stable transformation system for B. argenteum. Two homologues of ABI3(ABA-INSENSITIVE 3) gene were successfully cloned from B. argenteum. To further investigate the function of the ABI3 gene in B. argenteum, we used the CRISPR/Cas9 genetic editing system to target the BaABI3A and BaABI3B gene in B. argenteum protoplasts. This resulted in mutagenesis at the target in about 2-5% of the regenerated plants. The isolated abi3a and abi3b mutants exhibited increased sensitivity to desiccation, suggesting that BaABI3A and BaABI3B play redundant roles in desiccation stress. Overall, our results provide a rapid and simple approach for molecular genetics in B. argenteum. This study contributes to a better understanding of the molecular mechanisms of plant adaptation to extreme environmental.
建立银叶真藓的无菌培养体系和原生质体再生体系，并在银叶真藓中建立和应用CRISPR/Cas9系统。银叶真藓是一种迷人的、分布广泛且用途多样的苔藓物种，在各种受干扰的环境中都能茁壮成长。由于其对干燥、高紫外线和极端温度具有全面的耐受性，它正逐渐成为研究植物对非生物胁迫响应的分子机制的模式苔藓。然而，缺乏基因转化和靶向基因组编辑等基础工具阻碍了对银叶真藓在不同环境中生存的分子机制的理解。在此，我们报道银叶真藓的原丝体在失水高达95.4%时仍能存活。此外，通过kmer分析，银叶真藓的基因组大小约为313 Mb，小于先前报道的700 Mb。我们还开发了一种简单的原丝体诱导方法以及一种高效的银叶真藓原生质体分离和再生方案。此外，我们建立了一种聚乙二醇介导的银叶真藓原生质体瞬时转染和稳定转化系统。成功从银叶真藓中克隆出两个ABI3（脱落酸不敏感3）基因的同源物。为了进一步研究ABI3基因在银叶真藓中的功能，我们使用CRISPR/Cas9基因编辑系统在银叶真藓原生质体中靶向BaABI3A和BaABI3B基因。这导致约2-5%的再生植株在靶位点发生诱变。分离得到的abi3a和abi3b突变体对干燥的敏感性增加，表明BaABI3A和BaABI3B在干燥胁迫中发挥冗余作用。总体而言，我们的结果为银叶真藓的分子遗传学提供了一种快速简便的方法。本研究有助于更好地理解植物适应极端环境的分子机制。
