全 文 ：植物学报 Chinese Bulletin of Botany 2014, 49 (6): 643–652, www.chinbullbotany.com
doi: 10.3724/SP.J.1259.2014.00643
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收稿日期: 2013-09-24; 接受日期: 2014-06-02
基金项目: 国家自然科学基金(No.31230008, No.91017012)和中国博士后科学基金(No.2012T50080, No.2013T60102)
† 共同第一作者
* 通讯作者。E-mail: songss@tsinghua.edu.cn
拟南芥转录因子bHLH17负调控茉莉素介导的抗性反应
王翠丽1, 2†, 田海霞1, 2†, 汪姣姣2†, 齐天从2, 黄煌2, 任春梅1, 谢道昕2, 宋素胜2*
1湖南农业大学生物科学技术学院, 长沙 410128; 2清华大学生命科学学院, 清华北大生命科学联合中心, 北京 100084
摘要 植物激素茉莉素作为抗性信号调控植物对腐生性病原菌和昆虫的抗性, 作为发育信号调控植物根的生长、雄蕊发育、
表皮毛形成和叶片衰老。茉莉素受体COI1识别茉莉素分子, 进而与JAZ蛋白互作并诱导其降解, 继而调控多种茉莉素反应。
拟南芥(Arabidopsis thaliana) IIId亚组bHLH转录因子(bHLH3、bHLH13、bHLH14和bHLH17)是JAZ的一类靶蛋白。与野生
型相比, IIId亚组bHLH转录因子的单突变体对灰霉菌和甜菜夜蛾的抗性无明显差异, 而四突变体对灰霉菌和甜菜夜蛾的抗
性增强。该文通过高表达bHLH17并研究其对灰霉菌和甜菜夜蛾的抗性反应, 结果显示, 被灰霉菌侵染的bHLH17高表达植
株较野生型表现出更严重的病症。取食bHLH17高表达植株叶片的甜菜夜蛾幼虫体重大于取食野生型叶片的幼虫体重。
bHLH17高表达抑制了茉莉素诱导的抗性相关基因(Thi2.1)和伤害响应基因(VSP2、AOS、JAZ1、JAZ9和JAZ10)的表达。
原生质体转化实验显示bHLH17通过其N端行使转录抑制功能。研究结果表明, IIId亚组bHLH转录抑制因子bHLH17高表达
会负调控茉莉素介导的对灰霉菌和甜菜夜蛾的抗性。
关键词 bHLH17, COI1, 抗性, 茉莉素
王翠丽, 田海霞, 汪姣姣, 齐天从, 黄煌, 任春梅, 谢道昕, 宋素胜 (2014). 拟南芥转录因子bHLH17负调控茉莉素介导的
抗性反应. 植物学报 49, 643–652.
茉莉素是一种重要的植物激素 (Shan et al.,
2012; Wasternack and Hause, 2013)。它作为抗性信
号调控植物对腐生性病原真菌和昆虫的抗性
(McConn et al., 1997; Thomma et al., 1998; Yan et
al., 2013; Song et al., 2014); 调控植物对非生物胁
迫的抗性, 如紫外线(Fedina et al., 2009)、臭氧(Rao
et al., 2000)、冷害(Hu et al., 2013)和伤害(Mason
and Mullet, 1990; 宋恒和王长泉, 2013); 调控植物
的生长发育, 如根的生长(Feys et al., 1994)、表皮毛
发育(Yoshida et al., 2009; Qi et al., 2011, 2014)、雄
蕊发育(McConn and Browse, 1996; Song et al.,
2013b; Huang et al., 2014)和叶片衰老(Schommer
et al., 2008; Shan et al., 2011; Jiang et al., 2014);
调控次级代谢, 如花色素苷(Qi et al., 2011)、烟碱
(Zhang et al., 2012)、芥子油苷(Schweizer et al.,
2013)和倍半萜(Hong et al., 2012)的合成。
植物茉莉素受体CORONATINE INSENSITIVE1
(COI1)(Xie et al., 1998; Yan et al., 2009)识别活性茉
莉素分子茉莉酸异亮氨酸 (JA-Ile)(Fonseca et al.,
2009), 进而与一类抑制因子 JASMONATE-ZIM-
DOMAIN (JAZ)蛋白互作(Chini et al., 2007; Santner
and Estelle, 2007; Yan et al., 2007), 并泛素化JAZ
蛋白。泛素化的JAZ蛋白被26S蛋白酶体降解。在植
物体内, JAZ蛋白与下游的转录因子互作, 抑制植物
的茉莉素反应。当植物受到侵染、伤害, 或发育到一
定阶段时, 植物合成茉莉素分子。合成的茉莉素分子
通过COI1诱导JAZ蛋白降解, 进而释放出下游的转
录因子, 促进茉莉素反应。
JAZ蛋白与转录因子MYB21和MYB24互作特异
地调控雄蕊的发育(Song et al., 2011); 与WD-rep-
eat/bHLH/MYB复合体互作调控表皮毛发育和花色素
苷积累(Qi et al., 2011); 与MYC2、MYC3和MYC4互
作调控根的生长以及对昆虫和假单胞菌的抗性
(Cheng et al., 2011; Fernández-Calvo et al., 2011;
Niu et al., 2011); 与EIN3和EIL1互作调控对灰霉菌
的抗性(Zhu et al., 2011)。
·研究报告·
644 植物学报 49(6) 2014

拟南芥(Arabidopsis thaliana)茉莉素受体突变体
coi1由于JAZ蛋白的积累失去了对灰霉菌 (Botrytis
cinerea)等腐生性病原菌和甜菜夜蛾 (Spodoptera
exigua)等昆虫的抗性。EIN3和EIL1的双突变体丧失
了对灰霉菌的抗性, 表明EIN3和EIL1正调控茉莉素
介导的对灰霉菌的抗性。MYC2的突变体对昆虫的抗
性减弱, 但对灰霉菌的抗性增强, 表明MYC2正调控
茉莉素介导的对昆虫的抗性, 负调控茉莉素介导的对
灰霉菌的抗性。
拟南芥IIId亚组bHLH转录因子bHLH3、bHLH13、
bHLH14和 bHLH17与 JAZ蛋白互作 (Song et al.,
2013a)。与野生型相比, bHLH3、bHLH13、bHLH14
和bHLH17的单突变体对灰霉菌、假单胞菌和甜菜夜
蛾的抗性无明显变化 , 而四突变体bhlh3 bhlh13
bhlh14 bhlh17对灰霉菌和甜菜夜蛾幼虫的抗性增强,
对假单胞菌的抗性减弱(Song et al., 2013a)。这些研
究表明, IIId亚组bHLH转录因子参与茉莉素介导的对
灰霉菌、假单胞菌和甜菜夜蛾幼虫的抗性反应。
IIId亚组bHLH转录因子的高表达如何影响植株
对灰霉菌和甜菜夜蛾的抗性尚未见报道。本研究旨在
探讨高表达IIId亚组bHLH转录因子bHLH17的全长基
因对灰霉菌和甜菜夜蛾幼虫抗性的影响。灰霉菌活体
接种实验和离体接种实验显示, bHLH17超表达植株
较野生型对灰霉菌更敏感。昆虫取食实验显示 ,
bHLH17超表达植株对甜菜夜蛾幼虫更为敏感。实时
定量PCR分析结果显示, bHLH17超表达抑制了茉莉
素诱导的抗性相关基因 (Thi2.1)和伤害响应基因
(VSP2、AOS、JAZ1、JAZ9和JAZ10)的表达。进一
步的拟南芥原生质体转化实验表明, bHLH17通过N
端结构域行使转录抑制因子的功能。
1 材料与方法
1.1 植物材料
用20%八四消毒液浸泡拟南芥(Arabidopsis thaliana
L.)种子10分钟, 之后用灭菌双蒸水清洗5次。将种子
点播于MS培养基上, 在黑暗下低温(4°C)处理3天,
移到植物房9天, 再移栽到土壤中, 继续在植物房中
生长。在植物房或培养箱中进行光暗交替处理: 光照
16小时 , 温度为22–24°C; 黑暗8小时 , 温度为19–
22°C。
1.2 bHLH17超表达转基因植株的构建
通过PCR扩增(引物为acgcgtcgacatgaatatgagtgatttagg
和cggactagtttatatatcaccagagacctg)和限制性内切酶
(SalI和SpeI)酶切将bHLH17的CDS序列连接入改造
的pCAMBIA1300载体上。在该载体中bHLH17被35S
启动子驱动。通过农杆菌转化法将该载体转入拟南芥,
并获得多个独立的转基因株系。通过实时定量PCR检
测其超表达水平。对3个独立转基因株系的表型分析
显示这些株系表型一致。
1.3 灰霉菌侵染实验
对生长4周的拟南芥植株喷施悬浮于0.025%吐温20
的灰霉菌孢子悬浮液(浓度为107个 ·mL–1), 以喷施
0.025%吐温20溶液作为负对照。将喷施过溶液的拟
南芥植株放置于22°C黑暗处和高湿度(~100%)环境
下36小时, 之后转移到长日照(16小时光照/8小时黑
暗)且高湿度的培养箱中。侵染5天后统计叶片病症程
度(0: 无病症; 1: 弱病症; 2: 强病症; 3: 死亡)。每种
基因型取30个植株用于侵染实验。实验重复3次。
从生长4周的相应基因型拟南芥植株上取下8片
大小相似的叶片。将浓度为107个·mL–1灰霉菌孢子悬
浮溶液和对照溶液10 μL分别点接于叶片上。将叶片
置于铺有湿润滤纸、直径为150 mm的培养皿内, 密
封保湿。将培养皿放于22°C保存。侵染2天后计算病
斑直径。实验重复3次。随机选取相应基因型叶片进
行台盼蓝染色, 在显微镜下观察孢子萌发和菌丝生长
情况。
1.4 甜菜夜蛾幼虫取食实验
从生长4周的拟南芥植株上取下大小相似的叶片, 放
入铺有湿润滤纸、直径为90 mm的培养皿上。将甜菜
夜蛾虫卵置于27°C孵化。将新孵化的甜菜夜蛾幼虫经
饥饿处理12小时后, 放置于相应基因型叶片上取食。
每种基因型用5个培养皿进行重复实验。每2天更换1
次叶片。喂养6天后对幼虫进行称重。实验重复3次。
1.5 基因表达水平分析
拟南芥在培养皿中生长11天后, 经100 μmol·L–1茉莉
酸甲酯或对照溶液处理相应时间后 , 用于提取
RNA和后续的反转录实验。实时定量PCR分析利用
RealMasterMix(SYBR Green I)试剂盒 (Takara)和
王翠丽等: 拟南芥转录因子 bHLH17 负调控茉莉素介导的抗性反应 645

ABI7500实时定量PCR仪完成。以ACTIN8作内参
基因。检测各个基因的引物分别为: bHLH17(caga-
gaaaagaccagtgagcttg 和 gtctcttctcatcaacaacagaaa-
cta); Thi2.1(tgctcataatgagtctggtc和 tacaccttcccttgg-
aaaac); VSP2(tcagtgaccgttggaagttgtg和gttcgaacc-
attaggcttcaatatg); AOS(aactcctgatctaaccgtag和agt-
aatcccaacggtctttg); JAZ1(gagcaaaggcaccgctaata和
tgcgatagtagcgatgttgc); JAZ9(cgctcctaagcctaagttcca
和ttccgagcttgagggactg); JAZ10(cgctcctaagcctaagtt-
cca和tcgaaatcgcaccttgaata); ACTIN8(tcagcactttcc-
agcagatg和ctgtggacaatgcctggac)。
1.6 原生质体瞬时转化
转录活性检测采用基于GAL4 DNA结合结构域
(GAL4-DB)及其结合位点(GAL4(4x)-D1-3(4x))的瞬
时转化系统(Guo et al., 2013)。我们将bHLH17的
CDS 序 列 或 其 相 应 结 构 域 序 列 (bHLH17NT 和
bHLH17CT)与GAL4的DNA结合结构域融合。被
GAL4 DNA结合结构域结合位点GAL4(4x)-D1-3(4x)
驱动的GUS基因用作报告基因。35S启动子驱动的萤
火虫荧光素酶基因(firefly luciferase, LUC)用作内参。
拟南芥原生质体制备和转化方法参照相关文献(Yoo
et al., 2007)。GUS绝对活性与LUC绝对活性的比值
表示为GUS相对活性。载体构建的引物为: bHLH17-
DB(tcccccggggatgaatatgagtgatttaggttg 和 acgcgtcg-
acttatatatcaccagagacctgtg); bHLH17NT-DB(tccccc-
ggggatgaatatgagtgatttaggttg和acgcgtcgacttattcttgc-
tgagtgtttgtagatggattc); bHLH17CT-DB(tcccccgggg-
gaaaaatcagaatcttgtacagagaaa和acgcgtcgacttatata-
tcaccagagacctgtg)。
2 结果与讨论
2.1 bHLH17负调控拟南芥对腐生性病原菌灰霉
菌的抗性
为探讨高表达转录因子bHLH17对抗性的影响, 用
35S启动子驱动bHLH17的表达, 并通过农杆菌沾花
法获得bHLH17超表达转基因植株(图1A)。通过活体


图1 拟南芥bHLH17超表达植株对灰霉菌的抗性减弱
(A) 实时定量PCR检测生长11天的Col-0野生型(WT)和bHLH17超表达植株(bHLH17OE)幼苗中bHLH17基因的表达水平, 以
ACTIN8为内参; (B) 生长4周的野生型、bHLH17OE和茉莉素受体突变体(coi1-1)植株经灰霉菌侵染后第5天的叶片病症分级; (C) 生
长4周的野生型、bHLH17OE和coi1-1植株经灰霉菌侵染后第7天的植株表型

Figure 1 The Arabidopsis bHLH17 overexpression transgenic plants exhibited reduced resistance to Botrytis cinerea
(A) Quantitative real-time PCR analysis of expression level of bHLH17 in 11-day-old seedlings of Col-0 wild-type (WT) and
transgenic plants overexpressing bHLH17 (bHLH17OE), ACTIN8 was used as the internal control; (B) Classification of disease
symptoms of the leaves from WT, bHLH17OE, and coi1-1 5 days after spray inoculation; (C) Disease symptoms of WT,
bHLH17OE, and coi1-1 mutant 7 days after spray inoculation with mock (CK) or Botrytis cinerea in the growth condition with high
humidity
646 植物学报 49(6) 2014


图2 拟南芥bHLH17超表达植株离体叶片对灰霉菌的抗性减弱
(A) 生长4周的野生型、bHLH17超表达植株(bHLH17OE)和
coi1-1突变体植株的离体叶片在接种对照溶液(CK)或灰霉菌
(Botrytis cinerea)孢子悬浮液2天后的表型; 台盼蓝染色图片显
示相应基因型叶片上灰霉菌孢子萌发和菌丝生长情况; (B) 生
长4周的野生型、bHLH17超表达植株和coi1-1突变体植株的离
体叶片在接种灰霉菌孢子悬浮液2天后的菌斑直径。** 表示差
异极显著(P<0.01, t检验)

Figure 2 The detached leaves of Arabidopsis bHLH17 over-
expression transgenic plants exhibited reduced resistance to
Botrytis cinerea
(A) Detached rosette leaves from 4-week-old plants of Col-0
(WT), bHLH17OE, and coi1-1 were inoculated with mock (CK,
top panel) or Botrytis cinerea (as middle panel) spores for 2
days; Trypan Blue staining (bottom panel) showed the germi-
nation and growth of hyphae on the indicated leaves; (B) The
lesion diameter for the indicated genotype was shown. ** Stu-
dent’s t-test significance compared with WT plants (P<0.01)
接种实验比较Col-0野生型(WT)、bHLH17超表达植株
(bHLH17OE)和茉莉素受体突变体coi1-1对腐生性病
原菌灰霉菌的抗性反应。植株经灰霉菌孢子侵染5天
后的叶片病症统计结果显示, bHLH17OE和coi1-1均
比野生型表现出更严重的病症(图1B)。侵染后第7天,
bHLH17OE和coi1-1植株几乎全部死亡, 而野生型植
株仍有部分存活, 表现为含有少量绿色叶片和生长点
组织(图1C)。灰霉菌活体侵染实验表明, 与coi1-1相
似, bHLH17超表达转基因植株较野生型对灰霉菌更
敏感。
通过离体接种实验比较野生型、bHLH17OE和
coi1-1对灰霉菌的抗性反应。剪取生长4周的拟南芥叶
片, 并接种灰霉菌孢子。2天后, 可以观察到bHLH17OE
和coi1-1的叶片病斑较野生型的叶片病斑大(图2A)。
统计结果显示, 野生型、bHLH17OE和coi1-1的菌斑
直径分别为3.2、4.7和5.9 mm(图2B)。进一步对接种
2天后的叶片进行台盼蓝染色。结果显示, 与野生型
相比, bHLH17OE和coi1-1叶片上灰霉菌的孢子萌发
更好, 菌丝生长更茂盛(图2A)。
以上活体接种实验、离体接种实验和台盼蓝染色
实验均表明, 与coi1-1相似, bHLH17超表达植株对灰
霉菌更敏感, 即bHLH17负调控茉莉素介导的对灰霉
菌的抗性反应。台盼蓝染色实验显示, bHLH17可能通
过影响孢子萌发和菌丝生长而影响灰霉菌的致病力。
2.2 bHLH17负调控拟南芥对甜菜夜蛾的抗性
进一步通过甜菜夜蛾幼虫取食实验比较野生型、
bHLH17OE和coi1-1对昆虫的抗性反应。将新孵化且
经过饥饿处理的甜菜夜蛾幼虫分别置于生长4周的野
生型、bHLH17OE和coi1-1的叶片上进行取食, 观察
叶片被取食情况并称量甜菜夜蛾幼虫取食不同叶片
后的重量。bHLH17OE和coi1-1叶片较野生型叶片更
容易被甜菜夜蛾幼虫取食(图3A)。在取食6天后, 取食
bHLH17OE和coi1-1叶片的甜菜夜蛾幼虫较取食野
生型叶片的幼虫大(图3B)。取食野生型、bHLH17OE
和coi1-1叶片6天后的甜菜夜蛾幼虫体重分别为0.39、
0.71和1.29 mg(图3C)。甜菜夜蛾幼虫取食实验表明,
bHLH17OE和coi1-1对甜菜夜蛾幼虫的抗性较野生
型弱, 即bHLH17负调控茉莉素介导的对甜菜夜蛾的
抗性。

王翠丽等: 拟南芥转录因子 bHLH17 负调控茉莉素介导的抗性反应 647


图3 拟南芥bHLH17超表达植株对甜菜夜蛾幼虫的抗性减弱
(A) 甜菜夜蛾幼虫取食前(CK)和取食后(Spodoptera exigua)的
生长4周的相应基因型植株(WT、bHLH17OE和coi1-1)叶片; (B)
新孵化的甜菜夜蛾幼虫取食相应基因型叶片6天后的表型
(Bar=2 mm); (C) 新孵化的甜菜夜蛾幼虫在相应基因型叶片上
取食6天后的重量。**表示差异极显著(P<0.01, t检验)

Figure 3 Arabidopsis bHLH17 overexpression transgenic
plants exhibited reduced resistance to Spodoptera exigua
(A) Representative rosette leaves of wild-type (WT), bHLH17
overexpression transgenic plants (bHLH17OE), and coi1-1
before feeding (CK) or after feeding with Spodoptera exigua;
(B) Representative S. exigua larvae before feeding (0 d) or 6
days after feeding (6 d) with rosette leaves from WT,
bHLH17OE, and coi1-1 (Bar=2 mm); (C) Larval weight of S.
exigua 6 days after feeding with rosette leaves of WT,
bHLH17OE, and coi1-1. ** Student’s t-test significance com-
pared with WT plants (P<0.01)
2.3 bHLH17负调控茉莉素响应基因的表达
为进一步研究bHLH17高表达对茉莉素响应基因表达
的影响, 我们通过实时定量PCR检测了受茉莉素诱
导的抗性相关基因和伤害响应基因在野生型、
bHLH17OE和coi1-1植株中的表达情况。硫素蛋白基
因Thi2.1是抵抗病原菌侵害的重要基因, 受病原菌侵
染和茉莉素的诱导(Bohlmann et al., 1998)。该基因
的表达水平在一定程度上反映植株对病原菌的抗性
水平。如图4所示, bHLH17超表达植株的Thi2.1表达
水平较野生型植株低。茉莉素明显诱导野生型植株中
Thi2.1基因的表达, 而bHLH17超表达明显抑制了茉
莉素对Thi2.1表达的诱导(图4)。这与bHLH17抑制拟
南芥对病原菌灰霉菌的抗性是一致的。
营养贮存蛋白基因VSP2和茉莉素合成酶基因
AOS是受茉莉素诱导的伤害响应基因, 在抵抗昆虫
侵害过程中发挥重要作用(Park et al., 2002; Liu et
al., 2005)。这2个基因的表达水平在一定程度上可以
反映植株对昆虫侵害的抗性水平。如图4所示, VSP2
和AOS在bHLH17超表达植株中的表达水平低 , 且
bHLH17高表达抑制了茉莉素对VSP2和AOS的诱
导。据报道, 甜菜夜蛾幼虫取食和伤害都会诱导JAZ
基因 (JAZ1、JAZ9和JAZ10)的表达 (Chung et al.,
2008)。实时定量PCR分析结果显示, 茉莉素诱导野
生型植株中JAZ1、JAZ9和JAZ10的表达, 而bHLH17
高表达抑制茉莉素对JAZ1、JAZ9和JAZ10的诱导(图
4)。实时定量PCR分析结果表明, bHLH17高表达负调
控茉莉素响应基因的表达。
2.4 bHLH17通过其N端结构域行使转录抑制功能
我们进一步采用基于GAL4 DNA结合结构域(GAL4-
DB)及其结合位点(GAL4(4x)-D1-3(4x))的瞬时转化
系统(Guo et al., 2013)分析bHLH17是如何行使转录
功能的。GAL4-DB可以结合到特定的启动子序列上,
而融合GAL4-DB的蛋白可以激活或抑制启动子驱动
的下游报告基因GUS的表达。拟南芥原生质体瞬时转
化实验显示, 与对照相比, 融合GAL4-DB的bHLH17
抑制GUS的活性, 表明bHLH17作为转录抑制因子起
作用(图5A, C)(Song et al., 2013a)。
bHLH17的N端含有JID结构域(JAZ interaction
domain)(Fernández-Calvo et al., 2011), C端含有
648 植物学报 49(6) 2014


图4 拟南芥bHLH17超表达抑制茉莉素响应基因的表达
实时定量PCR分析茉莉素响应基因Thi2.1、VSP2、AOS、JAZ1、JAZ9和JAZ10在生长11天的相应基因型(WT和bHLH17OE)幼苗
中经对照(CK)或100 μmol·L–1茉莉酸甲酯(JA)处理6小时后的表达水平。内参基因为ACTIN8

Figure 4 Overexpression of Arabidopsis bHLH17 attenuated the expression of JA-responsive genes
Quantitative real-time PCR analysis of expression levels of Thi2.1, VSP2, AOS, JAZ1, JAZ9, and JAZ10 in 11-day-old seedlings
of WT and bHLH17OE treated with mock (CK) or 100 μmol·L–1MeJA (JA) for 6 hours. ACTIN8 was used as the internal control


bHLH结构域(图5B)。我们将bHLH17截短为含有JID
结构域的N端(bHLH17NT)和含有bHLH结构域的C
端, 并分别与GAL4-DB结构域融合, 检测其转录活
性。如图5C所示, bHLH17的N端仍具有转录抑制功
能, 而C端不具有。这表明bHLH17通过其N端结构域
行使转录抑制功能。
2.5 讨论
IIId亚组bHLH转录因子的单突变体对灰霉菌、假单胞
菌和甜菜夜蛾的侵害无明显表型, 而四突变体bhlh3
bhlh13 bhlh14 bhlh17对灰霉菌和甜菜夜蛾的抗性增
强, 对假单胞菌更敏感。bHLH17在融合了具有强转
录抑制功能的结构域SRDX之后, 经超表达可以抑制
拟南芥对甜菜夜蛾的抗性(Nakata et al., 2013)。本研
究通过高表达bHLH17全长发现bHLH17高表达抑制
茉莉素介导的对灰霉菌和甜菜夜蛾的抗性反应, 以及
茉莉素诱导的抗性基因的表达, 并揭示了bHLH17通
过N端结构域行使转录抑制功能。
研究进一步阐述了JAZ下游转录因子调控茉莉素
介导的对灰霉菌和昆虫抗性的机制模型: 在没有茉莉
素时, JAZ与EIN3/EIN1和MYC2/MYC3/MYC4结合
抑制其转录激活功能, 进而抑制植株对病原菌和昆虫
王翠丽等: 拟南芥转录因子 bHLH17 负调控茉莉素介导的抗性反应 649



图5 bHLH17通过N端结构域行使转录抑制功能
(A) 用于拟南芥原生质体瞬时转化实验的效应载体、报告载体和内参载体 ; (B) 截短的bHLH17N端 (bHLH17NT)和C端
(bHLH17CT)(数字表示结构域所含氨基酸范围; JID结构域和bHLH结构域分别被标示为灰色和黑色; bHLH17、bHLH17NT和
bHLH17CT分别与GAL4的DNA结合结构域(GAL4DB)融合); (C) 原生质体瞬时转化实验显示bHLH17全长和N端(bHLH17NT)具备
转录抑制功能

Figure 5 N-terminus is essential for the transcriptional repression function of bHLH17
(A) The schematic diagram shows the constructs used in the transient expression assays; (B) The schematic diagram shows the
bHLH17 domain constructs (The numbers indicate the positions of amino acid; The conserved JID domain and bHLH domain are
shown with grey and black box, respectively; Different bHLH17 domains were fused with GAL4 DNA binding domain (DB)); (C)
Transient expression assays show that the N-terminus is required for the transcriptional repression function of bHLH17

的抗性 , 也与 IIId亚组 bHLH转录因子 (bHLH3、
bHLH13、bHLH14和bHLH17)互作干扰其转录抑制
功能; 当植物受到病原菌侵染或昆虫侵害时, 植物合
成茉莉素, 茉莉素通过COI1诱导JAZ蛋白的降解, 释
放下游转录因子EIN3/EIN1和MYC2/MYC3/MYC4及
IIId亚组bHLH转录因子; 被释放的EIN3和EIL1促进
植株对腐生性病原菌的抗性, 而被释放的MYC2和
IIId亚组bHLH转录因子在一定程度上拮抗这种抗性,
进而避免过度的抗性反应; 被释放的MYC2/MYC3/
MYC4促进植株对昆虫的抗性反应, 而被释放的IIId
亚组bHLH转录因子抑制这种抗性反应。正向调节因
子和负向调节因子作为JAZ的靶蛋白同时存在, 并拮
抗地调控同一抗性反应, 有利于避免过度的抗性反
应, 促进植株适应不断变化的环境, 继而能够生存和
生长。
原生质体转化实验显示, IIId亚组bHLH转录因子
bHLH17通过其N端结构域行使转录抑制功能(图5),
而同属于第III组IIIe亚组bHLH转录因子MYC2通过其
N端TAD结构域行使转录激活功能 (Zhai et al.,
2013)。IIId亚组和IIIe亚组bHLH转录因子在N端结构
域上有很高的相似性(Toledo-Ortiz et al., 2003), 却
分别行使转录抑制和转录激活功能。该机制有待于进
一步验证。也有报道显示MYC2可以结合靶基因启动
子并抑制其表达(Chen et al., 2011)。至于IIId亚组
bHLH转录因子能否结合靶基因启动子并激活其表达
则需要进一步研究。
我们也注意到MYC2和bHLH17都负调控拟南芥
对灰霉菌的抗性(图1, 图2)(Lorenzo et al., 2004;
650 植物学报 49(6) 2014

Song et al., 2013a; Zhai et al., 2013)。bHLH17高表
达可以抑制拟南芥对灰霉菌的抗性(图1, 图2), 而
MYC2高表达却不能明显抑制拟南芥对灰霉菌的抗性
(Song et al., 2013a); bHLH17同时负调控茉莉素介
导的对腐生性病原菌和昆虫的抗性, 而MYC2负调控
茉莉素介导的对腐生性病原菌的抗性(Lorenzo et al.,
2004; Chen et al., 2011), 正调控茉莉素介导的对昆
虫的抗性(Fernández-Calvo et al., 2011)。今后更深
入地研究同属于第III亚组的IIId和IIIe亚组bHLH转录
因子将有助于解释这些科学问题。
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bHLH17 Negatively Regulates Jasmonate-mediated Plant
Defense Responses
Cuili Wang1, 2†, Haixia Tian1, 2†, Jiaojiao Wang2†, Tiancong Qi2, Huang Huang2, Chunmei Ren1,
Daoxin Xie2, Susheng Song2*
1College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China
2Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China
Abstract The plant hormone jasmonate (JA) functions as a defense signal to regulate plant defense against insect
attack and pathogen infection. It also acts as a developmental signal to regulate plant development and growth, including
root growth, stamen development, trichome initiation, and leaf senescence. The JA receptor CORONATINE INSENSI-
TIVE1 (COI1) perceives JA signals for interaction with JASMONATE-ZIM-DOMAIN (JAZ) proteins and subsequent ubiq-
uitination of JAZs, thereby regulating the JA responses. The single mutants of the bHLH subgroup IIId factors (bHLH3,
bHLH13, bHLH14 and bHLH17), which interact with JAZs, show no obvious phenotype in resistance to the fungus Botrytis
cinerea and defense against the herbivore Spodoptera exigua. However, the quadruple mutant shows enhanced resis-
tance to B. cinerea and defense against S. exigua. We investigated whether the overexpression of bHLH17 in Arabidopsis
affected the resistance to B. cinerea and defense against S. exigua. Similar to the JA receptor mutant coi1-1, Arabidopsis
bHLH17-overexpressing plants were more susceptible to B. cinerea and S. exigua than the wild type. As compared with
the wild type, bHLH17 overexpression attenuated the JA-inducible expression of defense- or wounding-related genes,
such as Thi2.1, VSP2, AOS, JAZ1, JAZ9 and JAZ10. Further transient expression analysis revealed that the N-terminus
of bHLH17 was essential for its transcriptional repression function. Overexpression of bHLH17 negatively regulates plant
defense against B. cinerea and S. exigua.
Key words bHLH17, COI1, defense, jasmonate
Wang CL, Tian HX, Wang JJ, Qi TC, Huang H, Ren CM, Xie DX, Song SS (2014). bHLH17 negatively regulates jas-
monate-mediated plant defense responses. Chin Bull Bot 49, 643–652.
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† These authors contributed equally to this paper
* Author for correspondence. E-mail: songss@tsinghua.edu.cn (责任编辑: 白羽红)