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作物学报 ›› 2020, Vol. 46 ›› Issue (9): 1322-1331.doi: 10.3724/SP.J.1006.2020.04008

• 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BjuB.KAN4基因调控类黄酮的途径

高国应1,2,3(), 伍小方1,2,3, 黄伟1,3, 周定港1,3, 张大为1,3, 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 / 经济作物遗传改良与综合利用湖南省重点实验室, 湖南湘潭 411201
  • 收稿日期:2020-01-12 接受日期:2020-04-15 出版日期:2020-09-12 网络出版日期:2020-04-27
  • 通讯作者: 张凯旋,严明理
  • 作者简介:E-mail: 1019982091@qq.com, Tel: 0731-58291416
  • 基金资助:
    本研究由国家重点研发计划项目(2016YFD0100202);国家自然科学基金项目(31971980);湖南省教育厅项目资助(17K035)

Regulation of flavonoid pathway by BjuB.KAN4 gene in Brassica juncea

GAO Guo-Ying1,2,3(), WU Xiao-Fang1,2,3, HUANG Wei1,3, ZHOU Ding-Gang1,3, ZHANG Da-Wei1,3, ZHOU Mei-Liang2, ZHANG Kai-Xuan2,*(), YAN Ming-Li1,3,*()   

  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 Hunan Key Laboratory of Economic Crops Genetic Improvement and Integrated Utilization, Hunan University of Science and Technology, Xiangtan 411201, Hunan, China
  • Received:2020-01-12 Accepted:2020-04-15 Published:2020-09-12 Published online:2020-04-27
  • Contact: Kai-Xuan ZHANG,Ming-Li YAN
  • Supported by:
    National Key Research and Development Program of China(2016YFD0100202);National Natural Science Foundation of China(31971980);Foundation of Hunan Education Department(17K035)

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摘要：

MYB类转录因子KAN4有调控植物原花青素合成的功能。为了探究芥菜型油菜中MYB转录因子KAN4对原花青素合成的调控机理, 本研究以芥菜型油菜紫叶芥为实验材料, 克隆了一个BjuB.KAN4基因, 编码266个氨基酸, BjuB.KAN4蛋白包含一段高度保守的MYB-like DNA结合结构域, 属于1R-MYB转录因子家族成员。基因表达分析表明, BjuB.KAN4在根中表达量显著高于叶和茎中, GUS组织化学染色分析试验推测, 该基因可能在根茎叶的维管组织中表达。利用毛状根体系过表达BjuB.KAN4发现, 类黄酮合成途径的部分关键酶基因Bju.CHS和Bju.DFR等的表达量在紫叶芥和四川黄籽的转基因根系中均显著增加, 紫叶芥转基因根系中总黄酮含量为2.798 mg g-1, 是对照组的1.3倍, 四川黄籽中总黄酮含量为2.567 mg g-1, 是对照组的1.2倍。在拟南芥中异源表达BjuB.KAN4发现, 转基因植株总黄酮含量为0.237 mg g-1, 是野生型的1.5倍, 原花青素含量为0.363 mg g-1, 较野生型含量下降。本研究表明, BjuB.KAN4基因参与调控芥菜型油菜类黄酮合成, 为研究芸薹属植物原花青素合成的调控机理提供了参考。

关键词: 芥菜型油菜, BjuB.KAN4, 类黄酮合成, 毛状根

Abstract:

MYB transcription factors KAN4 can effectively regulate the biosynthesis of plant proanthocyanidins. In order to investigate the function of the MYB transcription factor KAN4 on the regulation of proanthocyanidin synthesis in Brassica juncea, we cloned BjuB.KAN4 gene from purple-leaf mustard rape (PM), which encoded 266 amino acids. The BjuB.KAN4 protein contained a highly conserved MYB-like DNA-binding domain belongs to the 1R-MYB transcription factor family. BjuB.KAN4 gene expression showed significantly higher level in root than in leaf and stem. GUS histochemical staining showed that this gene might be expressed in vascular tissues. Overexpression of BjuB.KAN4 in hairy roots of PM and Sichuan Yellow (SY) increased the expression level of some key enzyme genes in the flavonoids biosythesis pathway, such as Bju.CHS and Bju.DFR. The total flavonoids content was 2.798 mg g-1 in transgenic roots of PM, which was 1.3 times higher than that of the control, and 2.567 mg g-1 in transgenic roots of SY, which was 1.2 times higher than that of control. In transgenic Arabidopsis plants overexpressing BjuB.KAN4, total flavonoids was 0.237 mg g-1, which was 1.5 times higher than that of wild type, however, the proanthocyanidins content decreased. This study indicates that the BjuB.KAN4 gene is involved in the regulation of PM flavonoid synthesis, and provides a reference for the research of regulation mechanism of proanthocyanidins synthesis in Brassica.

Key words: Brassica juncea, BjuB.KAN4, flavonoid synthesis, hairy roots

表1

引物序列"

引物名称
Primer name		 引物序列
Primer sequences (5°-3°)		 引物用途
Purpose of primer
KAN4-B-F AGTGAGATGATCATGTTCGAGTC 基因克隆Gene cloning
KAN4-B-R CAATTAGCACTTGAGAAGGGTTA
KB-NcoIF GGGGACTCTTGACCATGGTAATGATCATGTTCGAG KAN4载体构建引物
KAN4 vector primers
KB-Eco91IR GAAATTCGAGCTGGTCACCCAATTAGCACTTGAGAAGGG
KB-pro-BamHIF GGATCCATTGTCGTTGGTGACAGAAAC KAN4启动子载体构建引物
KB-pro vector primer
KB-pro-NcoIR CCATGGTTGGAGTTTTCAGAACTTTGGC
Actin7-F GCTGACCGTATGAGCAAAG qRT-PCR检测引物
qRT-PCR detection primers
Actin7-R AAGATGGATGGACCCGAC
qKAN4-B-F AGGACCCAAAGATCTCCTTGGT
qKAN4-B-R TTACACATAGTCAATCCCCCAACT
qPAL-F AGAGCTTTTGACCGGAGAGA
qPAL-R TTAATCACTCTTAACATATAGGAATGGGAG
qCHS-F TCTTCATATTGGACGAGATGAGGA
qCHS-R GCGTTTCTGTTCAAACAGGAA
qCHI-F CTTTGGAGCGACCATTAGAG
qCHI-R AGACAAAGCTTAACAAGAGAGGT
qF3’H-F TGATTGGGAATTAGCTGGAGGA
qF3’H-R AGTTAAATTTTAACCCGACCCGA
qF3H-F ATCTTGGAGGAGCCAATTACGT
qF3H-R ACACAAGGAGTCTAAGCGATGA
qFLS-F ACTAGGAATGTGATCGCACCA
qFLS-R TCAGAGGGATTAGGTTTACGG
qDFR-F TCTTTGGAACAGGTTTGAAGGA
qDFR-R TAAAGTGACAGGGAGAAAACCCT
qANS-F AAGCCGTTGCCTGAGA
qANS-R AGAGTTTCAGACTCAGACTTCA
qBAN-F GGTTTTTGTTGTTAGGGAAAGA
qBAN-R ATATGCTTACTCTGACAAAACAT

图1

BjuB.KAN4与其他物种同源KAN4蛋白的系统发育树 AtKAN4: 拟南芥NP199077; BjuB.KAN4芥菜型油菜; BolC.KAN4: 甘蓝XP013619450; BraA.KAN4: 白菜XP009123685; CpKAN4: 番木瓜XP021896463; CrKAN4: 荠菜XP006280924; CsKAN4: 亚麻芥XP010442142; EsKAN4: 山嵛菜XP006403368; GsKAN4: 野大豆XP028198852; PeKAN4: 胡杨XP011030894; RsKAN4: 萝卜XP018449685; ThKAN4: 醉蝶花XP010530376; VvKAN4: 葡萄CBI19594; ZmKAN4: 玉米NP001168849。"

图2

BjuB.KAN4基因的组织特异性表达分析 A: PM不同组织BjuB.KAN4的相对表达量; B: SY不同组织BjuB.KAN4的相对表达量。**表示在0.01水平差异显著。"

图3

芥菜型油菜毛状根的诱导过程和转基因根系鉴定 A: 芥菜型油菜毛状根的诱导过程。B: PM和SY转基因毛状根DNA鉴定; PKB1-PKB3: PM阳性毛状根; SKB1-SKB3: SY阳性毛状根; -: H2O (阴性对照); +: 质粒(阳性对照)。C: PM毛状根中BjuB.KAN4的相对表达量。D: SY毛状根中BjuB.KAN4的相对表达量。A4: A4侵染的毛状根; 3301: 转pCAMBIA3301空载体毛状根; KAN4-B1~B3: 转基因毛状根。**表示在0.01水平差异显著。"

图4

转基因毛状根中类黄酮代谢途径关键酶基因的相对表达量 A: PM转基因毛状根; B: SY转基因毛状根。A4: A4侵染的毛状根; 3301: 转pCAMBIA3301空载体毛状根; KAN4-B04: 转基因毛状根。内参基因: Actin7, 每组数据代表3次生物学重复的平均值±SD。PAL: 苯丙氨酸脱氨酶; CHS: 查尔酮合成酶; CHI: 查尔酮异构酶; F3H: 黄烷酮3-羟化酶; F3’H: 类黄酮3’-羟化酶; FLS: 黄酮醇合酶; DFR: 二氢黄酮醇还原酶; ANS: 花青素合成酶; BAN: 花色素还原酶。 *表示在0.05水平差异显著; **表示在0.01水平差异。"

图5

转基因毛状根中总黄酮含量检测 A: PM转基因毛状根; B: SY转基因毛状根。A4: A4侵染的毛状根; 3301: 转pCAMBIA3301空载体毛状根; KAN4-B04: 转基因毛状根。每组数据代表3次生物学重复的平均值±SD。*表示在0.05水平差异显著。"

图6

过表达BjuB.KAN4基因拟南芥分子鉴定 A: 转基因拟南芥阳性PCR鉴定; B: 转基因拟南芥中BjuB.KAN4的相对表达量检测。WT: 野生型拟南芥; KB-1-6: 转基因阳性株系; 3301: 转空载体拟南芥; KAN4-B1-3: 转BjuB.KAN4基因拟南芥。**表示在0.01水平差异显著。"

图7

转基因拟南芥总黄酮和原花青素含量检测 A: 转基因拟南芥总黄酮含量检测; B: 转基因拟南芥原花青素含量检测。WT: 野生型拟南芥; 3301: 转空载体拟南芥; KAN4-B04-1-3: 转BjuB.KAN4基因拟南芥; 每组数据代表3次生物学重复的平均值±SD。*表示在0.05水平差异显著; **表示在0.01水平差异显著。"

图8

转基因拟南芥组织化学检测 A, B, C: 转35Spro::GUS拟南芥的不同株系; D, E, F: 转BjuB.KAN4pro::GUS拟南芥的不同株系。"

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