棉纤维起始分化期基因枪介导的GhSuSy表达分析

doi:10.3969/j.issn.1004-1524.2022.07.02

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

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

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

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/Abstract

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

中图分类号:

S562

吕淑芳, 张红晓, 胥华伟, 赵杏利. 棉纤维起始分化期基因枪介导的GhSuSy表达分析[J]. 浙江农业学报, 2022, 34(7): 1361-1368.

LYU Shufang, ZHANG Hongxiao, XU Huawei, ZHAO Xingli. Analysis of GhSuSy expression by particle bombardment during initiation of cotton fibers[J]. Acta Agriculturae Zhejiangensis, 2022, 34(7): 1361-1368.

图/表 8

表1 qRT-PCR所用引物信息

Table 1 Information of qRT-PCR primers

基因Gene	上游引物Forward primer(5'→3')	下游引物Reverse primer(5'→3')	产物长度Production length/bp
GhUBQ7	GAAGGCATTCCACCTGACCAAC	CTTGACCTTCTTCTTCTTGTGCTTG	167
GhSuSy	TCGTAGTTGTAGGTGGTGATAGG	CACCGTTACAGGTTGCGAATG	275

图1 重组载体构建 A,GhSuSy启动子片段;B,瞬时表达激活载体。

Fig.1 Construction of recombinant vector A, Promoter fragment of GhSuSy; B, Transient expression vector.

表2 基因枪轰击参数对LUC表达活性的影响

Table 2 Effects of parameters of particle bombardment on activity of LUC

可裂膜压力 Pressure/ MPa	载体膜到叶片不同距离(cm)的LUC荧光强度 Luorescence intensity of luciferase of different target distance(cm)
可裂膜压力 Pressure/ MPa	5	7	9
4.60	(2.84±0.47) ×10³	(2.06±0.45) ×10³	(1.18±0.33) ×10³
6.80	(0.86±0.29) ×10⁵	(1.28±0.61) ×10⁵	(0.63±0.25) ×10⁵
9.20	(0.24±0.15) ×10³	(3.96±1.64) ×10³	(3.88±1.41) ×10³

图2 基因枪轰击次数对LUC表达效率的影响

Fig.2 Effects of the times of particle bombardment on LUC activity

图3 基因枪轰击后孵育时间对LUC活性的影响

Fig.3 Effects of incubating time on LUC activity

图4 纤维发育起始时期叶片和花瓣的相对LUC活性

Fig.4 Relative luciferase activity in leaves and petals during the initiation of fiber development

图5 纤维发育起始时期叶片、花瓣和胚珠中GhSuSy表达

Fig.5 Expression of GhSuSy in leaves, petals and ovules during initiation of fiber development

图6 纤维发育起始时期叶片、花瓣及胚珠的蔗糖合酶活性数据以鲜重计。

Fig.6 Sucrose synthase activity in leaves, petals and ovules during initiation of fiber development Data was detected based on fresh weight.

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棉纤维起始分化期基因枪介导的GhSuSy表达分析

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

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