生物技术通报 ›› 2025, Vol. 41 ›› Issue (9): 195-206.doi: 10.13560/j.cnki.biotech.bull.1985.2025-0231

• 研究报告 • 上一篇    

百合LoAPS1克隆及其在休眠解除过程的功能分析

徐小萍1(), 杨成龙1, 和兴1,2, 郭文杰1, 吴健3, 方少忠1()   

  1. 1.福建省农业科学院生物技术研究所 福建省农业遗传工程重点实验室,福州 350003
    2.福建农林大学农学院,福州 350002
    3.中国农业大学观赏园艺与园林学院 北京市观赏作物开发与质量控制重点实验室,北京 100193
  • 收稿日期:2025-03-05 出版日期:2025-09-26 发布日期:2025-09-24
  • 通讯作者: 方少忠,男,研究员,研究方向 :百合栽培生理与生物技术;E-mail: fangshaozhong@faas.cn
  • 作者简介:徐小萍,女,博士,助理研究员,研究方向 :观赏植物品质生理与生物技术;E-mail: byxxp310107@163.com
  • 基金资助:
    国家自然科学基金项目延伸研究项目(GJYS202407);福建省自然科学基金青年创新基金项目(2023J05064);国家自然科学基金青年项目(32302595)

Cloning of the LoAPS1 and Its Function Analysis during the Process of Dormancy Release in Lilium

XU Xiao-ping1(), YANG Cheng-long1, HE Xing1,2, GUO Wen-jie1, WU Jian3, FANG Shao-zhong1()   

  1. 1.Fujian Provincial Key Laboratory of Agricultural Genetic Engineering, Biotechnology Research Institute, Fujian Academy of Agricultural Science, Fuzhou 350003
    2.Institute of Agricultural College, Fujian Agriculture and Forestry University, Fuzhou 350002
    3.Beijing Key Laboratory of Development and Quality Control of Ornamental Crops, Department of Ornamental Horticulture, China Agricultural University, Beijing 100193
  • Received:2025-03-05 Published:2025-09-26 Online:2025-09-24

摘要:

目的 ATP硫酸化酶(APS1)是硫酸盐同化过程第一步的关键酶。研究百合鳞茎休眠解除过程APS1的作用,为理解硫代谢途径在百合鳞茎休眠解除中的功能提供参考。 方法 采用TA克隆从百合休眠解除不同阶段中获得LoAPS1基因序列全长,对其进行生物信息学分析,结合兰州百合基因组,分析APS1启动子顺式作用元件;利用百合休眠解除不同阶段及核黄素促进百合休眠解除的转录组数据库,分析APS1及硫代谢途径相关基因表达模式;采用烟草亚细胞定位瞬时转化技术验证LoAPS1蛋白的亚细胞定位;利用VIGS技术验证沉默LoAPS1对百合鳞茎休眠解除过程的作用。 结果 LoAPS1基因的ORF长为1 413 bp,GenBank登录号为WMQ58782.1,编码470个氨基酸;LoAPS1蛋白包含ATP-sulfurylase和PUA-like 2个保守结构域,亚细胞定位在叶绿体,可能与硫代谢通路APR、APK及SIR等发生互作;LoAPS1氨基酸序列与小果野蕉和姜亲缘性较高;百合APS1启动子包含4个转录起始位点,包含光响应元件、MYB结合位点及茉莉酸甲酯、赤霉素及脱落酸信号响应元件;LoAPS1及硫代谢通路相关基因在百合休眠解除过程下调表达;病毒介导的基因沉默技术及RT-qPCR结果表明,抑制LoAPS1表达促进百合鳞茎休眠解除。 结论 LoAPS1定位于叶绿体,可能响应茉莉酸甲酯、赤霉素及脱落酸等激素信号转导,抑制LoAPS1表达能够促进百合鳞茎休眠解除。

关键词: LoAPS1, 基因克隆, 病毒介导的基因沉默技术, 生物信息学分析, 亚细胞定位, 休眠解除, 百合

Abstract:

Objective ATP sulfurylase (APS1) is a key enzyme in the first step of the sulfate assimilation process. Studying the function of APS1 during dormancy release in Oriental Lilium ‘Siberia’ provides important theoretical guidance for understanding the function of sulphur metabolic pathways in dormancy release in lily bulbs. Method The full-length sequence of LoAPS1 gene was obtained from different stages of dormancy relase by TA cloning, and its bioinformatics analysis was performed. Combined with the Lanzhou lily genome, the cis-acting elements of APS1 promoter were analyzed. Using the transcriptome database of different phases of dormancy release in lily and the riboflavin promotion of dormancy release in lily, the expression patterns of APS1 genes and related genes of sulphur metabolism pathway were analyzed. The subcellular localization of LoAPS1 protein was verified by tobacco subcellular localization transient transformation technique. The effect of silencing LoAPS1 on the process of dormancy release in lily bulbs was verified by virus induced gene silencing (VIGS) technique. Result The LoAPS1 gene had an ORF length of 1 413 bp, GenBank accession number was WMQ58782.1, and encoded 470 amino acids. The LoAPS1 protein contained two conserved structural domains, ATP-sulfurylase and PUA-like, with subcellular localization in chloroplasts, and might interact with sulphur metabolism pathways, such as APR, APK and SIR. The amino acid sequence of LoAPS1 had a high affinity with Musa acuminata and Zingiber officinale. The lily APS1 promoter contained four transcriptional start sites, including light-responsive elements, MYB-binding sites, and signal-responsive elements for methyl jasmonate (MeJA), gibberellin (GA3), and abscisic acid (ABA). The expression of LoAPS1 and related genes of sulfur metabolism pathway were down-regulated during the process of dormancy release of lily. The results of the VIGS and RT-qPCR showed that inhibition of LoAPS1 expression promoted the dormancy release in lily bulbs. Conclusion LoAPS1 is localized in chloroplasts and may respond to hormone signaling such as MeJA, GA3 and ABA, and inhibition of LoAPS1 expression promotes the dormancy release in lily bulbs.

Key words: LoAPS1, gene cloning, VIGS, bioinformatics analysis, subcellular localization, dormancy release, Lilium