Abstract
Starch is the predominant compound in bulb scales, and previous studies have shown that bulblet development is closely associated with starch enrichment. However, how starch synthesis affects bulbification at the molecular level is unclear. In this study, we demonstrate that Lilium brownii var. giganteum, a wild lily with a giant bulb in nature, and L. brownii, the native species, have different starch levels and characteristics according to cytological and ultra-structural observations. We cloned the complete sequence of three key gene-encoding enzymes (LbgAGPS, LbgGBSS, and LbgSSIII) during starch synthesis by rapid amplification of 5′ and 3′ complementary DNA (cDNA) ends (RACE) technology. Bioinformatics analysis revealed that the proteins deduced by these genes contain the canonical conserved domains. Constructed phylogenetic trees confirmed the evolutionary relationships with proteins from other species, including monocotyledons and dicotyledons. The transcript levels of various tissues and time course samples obtained during bulblet development uncovered relatively high expression levels in bulblets and gradual increase expression accompanying bulblet growth. Moreover, a set of single nucleotide polymorphisms (SNPs) was discovered in the AGPS genes of four lily genotypes, and a purifying selection fashion was predicted according to the non-synonymous/synonymous (Ka/Ks) values. Taken together, our results suggested that key starch-synthesizing genes might play important roles in bulblet development and lead to distinctive phenotypes in bulblet size.
Abstract
目的
本研究基于鳞茎特异性膨大野生种质巨球百合与其原种野百合在淀粉含量及结构上的差异, 克隆并分析了巨球百合三个淀粉合成关键基因, 旨在为进一步深入研究淀粉代谢在鳞茎形成中的作用机制提供理论参考。
创新点
本研究首次从重要野生百合资源中系统克隆淀粉合成关键酶基因, 并分析其时空表达特性, 获得了AGPS基因候选单核苷酸多态性 (SNP) 位点。
方法
采用蒽酮法测定非结构性碳水化合物含量并用高碘酸-希夫反应染色法及投射电镜观察淀粉的显微及超微结构; 采用cDNA末端快速扩增 (RACE) 技术克隆获得巨球百合淀粉合成相关基因, 并应用BLAST, ExPASy, TMHMM, SWISS-MODE, Cell-PLoc, SMART, MEGA及KaKs_Calculator等生物信息学软件对其基因序列进行分析; 采用实时荧光定量PCR分析基因的时空表达差异。
结论
本研究证实巨球百合及原生种野百合在淀粉含量及特性方面存在差异, 并用RACE技术克隆获得了LbgAGPS, LbgGBSS及LbgSSIII基因。三种基因均在鳞茎中表达较高, 且随着鳞茎发育整体呈现上升趋势, 与鳞茎的形成存在较强的相关性。AGPS基因上存在丰富的SNP位点, 且主要为纯化选择。
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Acknowledgments
This work was supported by the National Natural Science Foundation of China (No. 31772337), the Lishui Research Fund (No. 2020zdhz03), the China Postdoctoral Science Foundation (No. 2017M612005), and the National Key Research and Development Program of China (No. 2018YFD10 00401). We gratefully thanks Tianyu HAN (Nanjing Forestry University, Nanjing, China) for his technical support.
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Yiping XIA, Yun WU, and Ziming REN designed the experiments, wrote and edited the manuscript. Minyi SUN, Shiqi LI, Ruihan MIN, Cong GAO, and Qundan LYU performed the study and data analysis. All authors have read and approved the final version of the manuscript and, therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data.
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Yun WU, Minyi SUN, Shiqi LI, Ruihan MIN, Cong GAO, Qundan LYU, Ziming REN, and Yiping XIA declare that they have no conflict of interest.
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Wu, Y., Sun, M., Li, S. et al. Molecular cloning, characterization and expression analysis of three key starch synthesis-related genes from the bulb of a rare lily germplasm, Lilium brownii var. giganteum. J. Zhejiang Univ. Sci. B 22, 476–491 (2021). https://doi.org/10.1631/jzus.B2000545
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DOI: https://doi.org/10.1631/jzus.B2000545