浙江农业学报 ›› 2022, Vol. 34 ›› Issue (7): 1361-1368.DOI: 10.3969/j.issn.1004-1524.2022.07.02

• 作物科学 • 上一篇    下一篇

棉纤维起始分化期基因枪介导的GhSuSy表达分析

吕淑芳(), 张红晓, 胥华伟, 赵杏利   

  1. 河南科技大学 农学院,河南 洛阳 471003
  • 收稿日期:2021-09-23 出版日期:2022-07-25 发布日期:2022-07-26
  • 作者简介:吕淑芳(1978—),女,河南许昌人,博士,讲师,主要从事植物生理与分子生物学研究。E-mail: Lvsf780515@haust.edu.cn
  • 基金资助:
    棉花生物学国家重点实验室开放课题(CB2018A21);棉花生物学国家重点实验室开放课题(CB2020A24);河南科技大学博士启动基金(13480086);河南省自然科学基金(212300410340)

Analysis of GhSuSy expression by particle bombardment during initiation of cotton fibers

LYU Shufang(), ZHANG Hongxiao, XU Huawei, ZHAO Xingli   

  1. College of Agriculture, Henan University of Science and Technology, Luoyang 471003, Henan, China
  • Received:2021-09-23 Online:2022-07-25 Published:2022-07-26

摘要:

蔗糖合酶(sucrose synthase)通过调节植物糖代谢介导棉纤维的起始和发育。为进一步研究棉纤维起始分化过程中蔗糖合酶的转录调控机制,克隆了GhSuSy基因5'端上游2 000 bp的启动子片段,构建了含LUC荧光报告基因的重组载体,采用基因枪瞬时转化技术,把重组载体DNA分别转化到棉花叶片和花瓣,比较分析了不同载体膜压力、不同轰击距离与孵育时间等条件下的荧光素酶(luciferase,LUC)活性。用优化后的条件检测了棉纤维起始分化期蔗糖合酶基因GhSuSy偶联的LUC在叶片和花瓣中的活性,并与GhSuSy表达、蔗糖合酶活性比较分析。结果显示:-0.09 MPa真空度下,可裂膜压力设为6.80 MPa,载体膜距离叶片7 cm,轰击2次转化效率最高,转化后样品孵育16 h左右LUC荧光信号最强;与叶片中LUC表达相比,花瓣中LUC荧光信号强、活性高,特别是开花当天(0 d)和开花后1 d(1 DPA)差异明显;在纤维起始分化的开花前3 d(-3 DPA)到开花后3 d(3 DPA),叶片和花瓣中LUC活性先升高后降低,在0和1 DPA的LUC活性最高,这一结果与GhSuSy的定量分析、蔗糖合酶活性检测结果趋于一致,同时纤维起始时期胚珠中GhSuSy的表达和蔗糖合酶活性也显著升高。这些结果暗示GhSuSy基因或编码的蔗糖合酶在0和1 DPA可能调控了棉纤维的起始和分化,表明基因枪介导的瞬时转化系统可用于进一步的GhSuSy基因启动子转录调控分析。

关键词: 棉纤维, GhSuSy基因, 蔗糖合酶, 基因枪瞬时表达

Abstract:

Sucrose synthase mediated the initiation and development of cotton fiber through regulating plant sugar metabolism. In order to further research the transcription mechanism of sucrose synthase in the process of the cotton fiber initial, the 2 000 bp promoter fragment of GhSuSy gene upstream was cloned and recombinant vector with luciferase (LUC) fluorescent reporter gene was constructed. The recombinant vector DNA were transformed into cotton leaves and petals by particle bombardment, respectively, and LUC activity were compared and analyzed under different pressure, different bombarding distance and incubation time.LUC activity in leaves and petals were determined at the initial differentiation stage of cotton fibers and were compared with GhSuSy expression and sucrose synthase activity. The results showed that the conversion efficiency was the highest under vacuum degree of -0.09 MPa, the pressure was set as 6.80 MPa, the distance of carrier film from the blade was 7 cm after two times of bombardment, LUC fluorescence was the strongest after the transformed samples were incubated for 16 h. Compared with LUC in leaf, LUC fluorescence in petal were stronger and activity were higher, especially at 0 and 1 DPA(days post anthesis).From 3 days before flowering (-3 DPA) to 3 days after flowering (3 DPA), the LUC activity in leaves and petals increased first and then decreased, and the LUC activity reached the highest at 0 and 1 DPA, which were consistent with the quantitative analysis of GhSuSy and sucrose synthase activity in leaves, petals and ovules. These results suggested that GhSuSy gene or the sucrose synthase significantly regulated the initiation and differentiation of cotton fibers at 0 and 1 DPA, and showed that gene gun-mediated transient transformation system could be used to analyze the transcriptional regulation of GhSuSy gene promoters.

Key words: cotton fiber, GhSuSy gene, sucrose synthase, particle bombardment

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