植物学报 ›› 2017, Vol. 52 ›› Issue (6): 713-722.doi: 10.11983/CBB16239

• 研究报告 • 上一篇    下一篇

甘蓝型油菜BnTTG1-1基因的功能分析

刘凯歌, 齐双慧, 段绍伟, 李东, 金倡宇, 高晨浩, 刘绚霞, 陈明训*()   

  1. 西北农林科技大学农学院, 杨凌 712100
  • 收稿日期:2016-12-05 接受日期:2017-04-03 出版日期:2017-11-01 发布日期:2017-11-22
  • 通讯作者: 陈明训 E-mail:cmx786@nwafu.edu.cn
  • 基金资助:
    国家自然科学基金(No.31501336)、西北农林科技大学引进人才启动经费(No.Z111021402)和优青培养科研专项(No. Z109021517)

Functional Analysis of Brassica napus BnTTG1-1 Gene

Liu Kaige, Qi Shuanghui, Duan Shaowei, Li Dong, Jin Changyu, Gao Chenhao, Liu Mingxun Chen Xuanxia*()   

  1. College of Agronomy, Northwest A&F University, Yangling 712100, China
  • Received:2016-12-05 Accepted:2017-04-03 Online:2017-11-01 Published:2017-11-22
  • Contact: Liu Mingxun Chen Xuanxia E-mail:cmx786@nwafu.edu.cn

摘要:

拟南芥(Arabidopsis thaliana) AtTTG1作为WD40重复转录因子存在于细胞核中, 对表皮毛形成、花青素合成和储藏物质积累等具有重要调节作用。该研究从甘蓝型油菜(Brassica napus)品种秦优7号中克隆获得了BnTTG1-1基因的全长CDS序列, 对其进行了烟草(Nicotiana benthamiana)叶片细胞的亚细胞定位研究, 检测了BnTTG1-1在油菜(B. campestris)中的时空表达模式, 并比较分析了BnTTG1-1对多个生物学过程的影响作用。结果表明, BnTTG1-1定位于烟草叶片细胞的细胞核中, 推测其作为转录因子发挥调节作用。BnTTG1-1广泛存在于油菜营养组织和发育的种子中。在突变体ttg1-13背景下, 异源表达BnTTG1-1基因能够完全恢复该突变体的多个表型, 如无表皮毛形成和花青素合成、种皮呈黄色、种子脂肪酸和储藏蛋白含量高以及在种子萌发和幼苗形态建成过程中对高葡萄糖和高盐胁迫耐受力差等。由此可知, 甘蓝型油菜BnTTG1-1与拟南芥AtTTG1在植物生长发育的多个生物学过程中具有类似的功能。

关键词: TTG1, 甘蓝型油菜, 拟南芥, 功能互补

Abstract:

AtTTG1 existing in the nucleus as a WD40 repeat transcription factor plays important roles in regulating trichome initiation, anthocyanin biosynthesis, and storage reserve accumulation in Arabidopsis thaliana. In the present study, we cloned the full-length coding domain sequence (CDS) of the BnTTG1-1 gene from the Brassica napus cv. ‘QINYOU Seven’, analyzed its subcellular localization, detected its temporal and spatial expression patterns in different tissues, and investigated its functions in several biological processes. BnTTG1-1 was localized in the nucleus of tobacco leaf cells, so it may function as a transcription factor. BnTTG1-1 was widely expressed in various vegetative tissues and developing seeds in QINYOU Seven. Moreover, introducing 35S:BnTTG1-1 into the mutant ttg1-13 fully rescued many phenotypes of the mutant, such as no trichomes and anthocyanins, yellow seed coat, higher contents of seed fatty acids and storage proteins, and sensitivity to higher sucrose or salinity stresses during seed germination and seedling establishment. Thus, BnTTG1-1 and AtTTG1 exhibited conserved functions on many biological processes during plant growth and development.

Key words: TTG1, Brassica napus, Arabidopsis thaliana, functional complementation

表1

引物序列"

Primer name Primer sequence (5′-3′) Annotation
AtACTIN7-F GCCCCTGAGGAGCACCCAGTT RT-PCR
AtACTIN7-R CCGGTTGTACGACCACTGGCA
BnTTG1-1-F GCCAGTATCCGTCCTCAACA RT-PCR
BnTTG1-1-R CTCCCAGATAAGAGCCTGCG
BnACTIN7-F GGAGCTGAGAGATTCCGTTG qRT-PCR
BnACTIN7-R GAACCACCACTGAGGACGAT
BnTTG1-1-F CTGCAGTGGTCTTCTTCGTT qRT-PCR
BnTTG1-1-R GTTACAATCACATAGATGCAGAGAC
BnTTG1-1-Xma1-F TATTcccgggATGGACAACTCAGCTCCAGACTC 35S:BnTTG1-1-GFP and 35S:BnTTG1-1
BnTTG1-1-Spe1-R GGactagtAACTCTAAGGAGCTGCATTTTGTTAGC

图1

qRT-PCR分析BnTTG1-1基因在甘蓝型油菜秦优7号种子不同发育时期的表达模式(平均值±标准差)DAP: 授粉后的天数。BnACTIN7为内参基因。"

图2

BnTTG1-1在烟草叶片细胞中的亚细胞定位DAPI: 4', 6-二脒基-2-苯基吲哚; GFP: 绿色荧光蛋白; Merge: DAPI、GFP和亮场3个图像的合并图像。Bars=5 μm"

图3

ttg1-13 35S:BnTTG1-1转基因植株的鉴定(A) 在DNA和RNA水平鉴定ttg1-13 35S:BnTTG1-1转基因植株, AtACTIN7为内参基因; (B) 在突变体ttg1-13背景下异源表达BnTTG1-1能够完全恢复突变体的表型, 如无表皮毛和花青素等。"

图4

比较拟南芥野生型(Col-0)、突变体ttg1-13和转基因植株ttg1-13 35S:BnTTG1-1的种皮颜色、种子大小和重量(平均值±标准差)(A) 成熟种子的显微观察; (B) 成熟种子的大小和重量比较"

图5

比较拟南芥野生型(Col-0)、突变体ttg1-13和转基因植株ttg1-13 35S:BnTTG1-1种子的储藏蛋白与脂肪酸含量(平均值±标准差)(A) 种子储藏蛋白含量; (B) 种子脂肪酸含量。*表示在P<0.05水平上差异显著。"

图6

非生物胁迫条件下(含有3%葡萄糖和100 mmol·L-1 NaCl)拟南芥野生型Col-0、突变体ttg1-13和转基因植株ttg1-13 35S: BnTTG1-1的发芽率和幼苗形态建成(A) 种子发芽率; (B) 幼苗的形态建成。数据为3个生物学重复的平均值±标准差, 每个生物学重复统计100粒种子。"

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