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作物学报 ›› 2025, Vol. 51 ›› Issue (3): 667-675.doi: 10.3724/SP.J.1006.2025.44132

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

生长素响应报告基因转化甘蓝型油菜及各组织GUS动态信号分析

张琴(), 戴成, 马朝芝()   

  1. 华中农业大学作物遗传改良全国重点实验室, 湖北武汉 430070
  • 收稿日期:2024-08-19 接受日期:2024-10-25 出版日期:2025-03-12 网络出版日期:2024-11-12
  • 通讯作者: *马朝芝, E-mail: yuanbeauty@mail.hzau.edu.cn
  • 作者简介:E-mail: 1963480575@qq.com
  • 基金资助:
    国家自然科学基金项目(32072105)

Auxin response reporter gene transformation of Brassica napus and dynamic signal analysis of GUS in different tissues

ZHANG Qin(), DAI Cheng, MA Chao-Zhi()   

  1. National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, Hubei, China
  • Received:2024-08-19 Accepted:2024-10-25 Published:2025-03-12 Published online:2024-11-12
  • Contact: *E-mail: yuanbeauty@mail.hzau.edu.cn
  • Supported by:
    National Natural Science Foundation of China(32072105)

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摘要：

为探究甘蓝型油菜中生长素在不同组织中的动态分布情况, 构建了以DR5::GUS为报告基因的生长素响应表达载体, 将其转化到甘蓝型油菜中, 获得了稳定表达GUS基因的转基因株系。GUS染色发现, 在油菜苗期, 子叶和下胚轴中都有比较强的GUS信号, 并且DR5启动子受生长素类似物NAA诱导, 而真叶和根部的GUS信号比较弱, 证明子叶中生长素含量比较高; 在油菜花蕾期, 花药以及萼片中有比较强的GUS信号, 而柱头中GUS信号比较弱, 说明生长素可能对花药的发育有比较重要的作用; 生长素在授粉后不同发育时期的种子以及角果中均表现出先升高再降低的趋势, 表明生长素可能在种子发育中发挥作用。综上所述, 本研究通过DR5::GUS生长素报告系统对甘蓝型油菜中的生长素进行可视化, 为进一步揭示生长素在油菜生长发育中的作用机制提供了新的技术手段。

关键词: 甘蓝型油菜, 生长素, DR5, 生长素分布

Abstract:

To investigate the dynamic distribution of auxin in various tissues of Brassica napus, a growth hormone-responsive expression vector with DR5::GUS as the reporter gene was constructed and transformed into B. napus. Transgenic lines stably expressing the GUS gene were obtained. GUS staining revealed that during the seedling stage, strong GUS signals were observed in the cotyledons and hypocotyl, while weaker signals were detected in the true leaves and roots, indicating higher auxin accumulation in the cotyledons. The DR5 promoter was also induced by the auxin analog NAA. At the bud stage, strong GUS signals were found in the anthers and sepals, with weaker signals in the stigma, suggesting that auxin may play a significant role in anther development. In seeds and siliques at various developmental stages after pollination, auxin levels exhibited an increase followed by a decrease, implying a role for auxin in seed development. In conclusion, this study visualized auxin distribution in B. napus using the DR5::GUS auxin reporter system, providing a valuable method for further elucidating the role of auxin in the growth and development of B. napus.

Key words: Brassica napus, auxin, DR5, auxin distribution

图1

生长素响应报告基因载体pc2300-DR5::GUS结构图"

表1

阳性转基因植株鉴定引物序列"

名称Name 序列Sequence (5′-3′)
正向引物Forward primer CTTGGATCCAAGCTTCCGACAC
反向引物Reverse primer TCTGCCAGTTCAGTTCGTTGTTCAC

表2

普通PCR体系"

组分Component 使用量Volume (μL)
正向引物Forward Primer 0.5
反向引物Reverse Primer 0.5
DNA template 1.0
2×Taq Plus Master Mix II (Dye plus) 5.0
ddH2O 3.0
合计Total 10.0

图2

GUS阳性植株的PCR鉴定以及GUS染色鉴定 A: 具有卡那霉素抗性的转基因油菜植株的PCR鉴定。M: marker, 目的条带大小为705 bp。No.1~8为8个转基因单株, 9为阳性对照, 10为阴性对照。B: 具有卡那霉素抗性的转基因油菜植株的GUS染色鉴定。"

图3

DR5::GUS转基因油菜幼苗在不同发育阶段GUS活性染色 A, C, E~I: 萌发1~15 d幼苗的GUS染色鉴定。B, D: 萌发1 d和2 d幼苗根尖的GUS染色鉴定。图E~I标尺: 1 cm。"

图4

DR5::GUS转基因油菜幼苗在不同发育阶段GUS与GH3.6基因表达量检测 使用t检验进行显著性分析(n ≥ 3), *和**分别表示在0.05和0.01水平差异显著。"

图5

不同浓度NAA处理下DR5::GUS转基因油菜幼苗GUS染色 标尺: 1 cm。"

图6

DR5::GUS转基因油菜在花蕾中的GUS染色 标尺: 100 μm。Bud: 花蕾; Stigma: 柱头; Sepal: 萼片; Anthers: 花药。Bar: 100 μm."

图7

DR5:: GUS转基因油菜在种子中的GUS染色 A~J: 授粉后1~20 d种子的GUS染色鉴定。标尺: 500 μm。"

图8

DR5::GUS转基因油菜在角果中的GUS染色"

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