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作物学报 ›› 2018, Vol. 44 ›› Issue (10): 1468-1476.doi: 10.3724/SP.J.1006.2018.01468

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

芥菜型油菜毛状根诱导体系构建及TTG1基因功能初步研究

李隆1,2,程成1,2,伍小方1,2,张大为1,3,刘丽莉1,3,周静1,周美亮2,张凯旋2,*(),严明理1,3,*()   

  1. 1湖南科技大学生命科学学院, 湖南湘潭 411201
    2中国农业科学院作物科学研究所, 北京 100081
    3湖南科技大学重金属污染土壤生态修复与安全利用湖南省普通高等学校重点实验室, 湖南湘潭 411201
  • 收稿日期:2018-02-14 接受日期:2018-06-12 出版日期:2018-10-10 网络出版日期:2018-07-02
  • 通讯作者: 张凯旋,严明理
  • 基金资助:
    本研究由国家重点研发计划项目(2016YFD0100202);湖南省自然科学基金项目(2016JJ1010);国家自然科学基金项目(31572457);湖南省教育厅项目(17K035)

Construction of Hairy Root Induction System and Functional Analysis of TTG1 Gene in Brassica juncea

Long LI1,2,Cheng CHENG1,2,Xiao-Fang WU1,2,Da-Wei ZHANG1,3,Li-Li LIU1,3,Jing ZHOU1,Mei-Liang ZHOU2,Kai-Xuan ZHANG2,*(),Ming-Li YAN1,3,*()   

  1. 1 College of Life Science, Hunan University of Science and Technology, Xiangtan 411201, Hunan, China
    2 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
    3 Key Laboratory of Ecological Remediation and Safe Utilization of Heavy Metal-Polluted Soils, Hunan University of Science and Technology, Xiangtan 411201, Hunan, China
  • Received:2018-02-14 Accepted:2018-06-12 Published:2018-10-10 Published online:2018-07-02
  • Contact: Kai-Xuan ZHANG,Ming-Li YAN
  • Supported by:
    This study was supported by the National Key Research and Development Program(2016YFD0100202);the Natural Science Foundation of Hunan Province(2016JJ1010);the National Natural Science Foundation of China(31572457);the Foundation of Hunan Education Department(17K035)

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摘要：

黄籽油菜具有种皮薄、出油率高等优点, 研究油菜黄籽的形成具有重要的意义。前期研究表明, TTG1 (TRANSPARENT TESTA GLABRA 1)基因参与了油菜种皮颜色的形成。本研究利用发根农杆菌A4菌株诱导了芥菜型油菜四川黄籽的毛状根, 并研究了菌液浓度和外植体部位对诱导率的影响。结果表明, 菌液浓度OD值为0.8时, 诱导效率最高, 平均为71.5%; 外植体中下胚轴的发根率最高, 平均为87.3%。在四川黄籽毛状根中过表达TTG1基因, 发现原花色素合成途径中的DFR (dihydroflavonol4-reductase)、ANS (anthocyanidin synthase)和BAN (anthocyanidin reductase)基因的表达受到抑制。本研究优化了油菜毛状根的诱导体系, 为利用毛状根体系进行基因功能验证提供了新的思路和方法。

关键词: 芥菜型油菜, 毛状根, 诱导条件, TTG1基因, 原花色素

Abstract:

Yellow seed rape has the advantages of thin seed coat and high oil yield. It is of great significance to study the formation of yellow seed in rapeseed. Previous studies showed that TTG1 (TRANSPARENT TESTA GLABRA 1) gene was involved in the formation of seed coat color. In this study, Agrobacterium rhizogenes A4 strain was used to induce hairy roots of Sichuan yellow, and investigate the effects of bacterial concentration and different sources of explants on hairy root induction. The highest average rooting rate was 71.5% when the OD value of A. rhizogenes A4 reached to 0.8. The average rooting rate of hypocotyls was significantly higher than that of cotyledons and up to 87.3% after the infection. Overexpression of TTG1 gene in Sichuan yellow hairy roots inhibited the expression of DFR (Dihydroflavonol4-reductase), ANS (Anthocyanidin synthase), and BAN (Anthocyanidin reductase) genes, encoding key enzymes of the proanthocyanidins biosynthesis. In this study, we optimized the induction system of hairy roots in B. juncea, and analyzed the function of TTG1 in the regulation of proanthocyanidins synthesis pathway, which provide a new idea and method for the functional analysis of genes in the hairy root system.

Key words: Brassica juncea, hairy root, inducing condition, TTG1 gene, proanthocyanidins

表1

qRT-PCR引物表"

基因
Gene		 名称
Name		 引物
Primer (5°-3°)		 PCR长度
PCR length (bp)
BAN Anthocyanidin reductase F: ATCTTCCATGTCGCAACTCC
R: CGAGAGAGATAAACTGCTAG		 200
DFR Dihydroflavonol4-reductase F: AGGATTCATTGGTTCATGGCTCG
R: TCCGTTTATGGCGTCATCGTAGC		 189
ANS Anthocyanidin synthase F: GGACAACTTGAGTGGGAAGATTA
R: GCAAACGAAGACACTTAGCGTAC		 132
TTG1 protein (“WD40” or “WDR”) F: GGCTGTAAGCATATCTGTTCCG
R: GAGAAAGCAATCCCAATCCAATG		 173
内参
Internal reference		 Actin7 F: GCTGACCGTATGAGCAAAG
R: AAGATGGATGGACCCGAC		 182

表2

不同外植体对油菜毛状根诱导率的影响"

外植体
Explant		 感染外植体数量
Number of infected explants		 外植体发根数量
Number of explants rooted		 诱导率
Induction rate (%)
下胚轴 Hypocotyl 30 26.2±0.3 a 87.3±0.01 a
子叶 Cotyledon 30 12.0±0.5 b 40.0±0.02 b

图1

毛状根的诱导过程 A: 14 d的四川黄籽油菜幼苗; B: 诱导7 d发根的外植体; C: 14 d以后的毛状根; D: 21 d以后转移到筛选培养基;E: 28 d在液体培养基上; F: 42 d以后的毛状根。"

图2

不同浓度菌液对毛状根的诱导效率"

图3

载体构建图 A: pCAMBIA3301载体; B: 改造后的载体。"

图4

转化菌液的检测 A: 35S-TTG1阳性检测结果。+: pCAMBIA3301-TTG1, -: H2O; B: GUS 阳性检测结果。+: pCAMBIA3301质粒, -: H2O。"

图5

转基因毛状根的鉴定 A: 转GUS基因的3个根系Z1、Z2、Z3的PCR阳性检测。+: pCAMBIA3301质粒; -: 野生型毛状根; B: 转TTG1基因的3个根系T1、 T2、T3的PCR阳性检测。+: pCAMBIA3301-TTG1; -: 野生型毛状根。"

图6

野生型和转GUS基因毛状根的染色结果 L1、L2、L3: 野生型的3个根系; Z1、Z2、Z3: 含有GUS基因的3个根系。"

图7

野生型和转基因毛状根生长状况 A1: 野生型毛状根根尖转入0 d状态; B1: 转GUS基因毛状根根尖0 d状态; C1: 转TTG1毛状根根尖0 d状态; A2: 野生型毛状根转入14 d状态; B2: 转GUS基因毛状根14 d状态; C2: 转TTG1毛状根14 d状态。"

图8

qRT-PCR检测相关基因的表达量 A: TTG1基因; B: DFR基因; C: ANS基因; D: BAN基因。"

图9

拟南芥原花青素代谢途径中TT2、TT8和TTG1形成MBW复合体调控DFR、ANS和BAN的示意图 PA代谢途径的酶基因分别为查尔酮合酶(CHS)、查尔酮异构酶(CHI)、黄烷酮3-羟化酶基因(F3H)、类黄酮3’-羟化酶基因(F3’H)、二氢黄酮醇4-还原酶(DFR)、花青素合成酶(ANS)、花色素还原酶(BAN)、谷胱甘肽S-转移酶(TT12)和MATE次级转运子(TT19)。"

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