植物非生物逆境胁迫DREBCBF转录因子的研究进展

第47卷第1期2011年1月

林

业

科

学

SCIENTIA

SILVAE

SINICAE

Vol.47,No.1Jan.,2011

植物非生物逆境胁迫DREB /CBF

转录因子的研究进展

李科友1　朱海兰2

(1.西北农林科技大学生命科学学院　杨凌712100;

2.西北农林科技大学林学院　杨凌712100)

摘　要:　综述近十几年来,特别是近5年来国内外DREB /CBF 转录因子的研究进展,主要包括DREB /CBF 转录因子的结构特点,DREB /CBF 基因的克隆、表达调控及其在植物抗逆基因工程中的作用,以及DREB /CBF 转录因子研究中的问题与展望,旨在为植物抗逆育种提供理论依据。DREB /CBF 类转录因子即干旱应答元件结合蛋白质/C -重复序列结合子,是AP2/EREBP 转录因子家族的一个亚家族,拥有保守的AP2结构域,能够特异性地与抗逆基

因启动子区域的DRE /CRT 顺式作用元件相结合,在干旱、低温和高盐等条件下调节一系列下游逆境应答基因的表达,是植物逆境适应中的关键性调节因子。目前,已从拟南芥、欧洲油菜、水稻、玉米、小麦、陆地棉、大豆和番茄等几十种植物中分离并鉴定出调控干旱、高盐及低温耐性的DREB /CBF 基因,并利用这些基因得到抗逆性增强的拟南芥、欧洲油菜、番茄、小麦以及杨树等转基因植株。转基因结果表明,DREB /CBF 转录因子家族在植物抗逆品种改良中具有重要的应用价值。

关键词:　DREB /CBF;转录因子;DRE /CRT 顺式作用元件;转基因植物;抗逆育种

中图分类号:S718.46;Q943.2 文献标识码:A 文章编号:1001-7488(2011)01-0124-11

收稿日期:2010-08-02;修回日期:2010-10-06。

Research Progress of DREB /CBF Transcription Factor in

Response to Abiotic?Stresses in Plants

Li Keyou 1　Zhu Hailan 2

(1.College of Life Sciences ,Northwest A &F University Yangling 712100;

2.College of Forestry ,Northwest A &F University Yangling 712100)

Abstract :　The research progress on DREB /CBF (Dehydration Responsive Element Binding Protein /C?repeat Binding Factor)transcription factors in last decades especially in recent five years is reviewed,mainly including the structural features of DREB /CBF transcription factors,the cloned DREB /CBF genes and their expression regulations,the application

of DREB /CBF genes in gene engenieering for improving plant stress resistance,as well as the existing problems and the future prospective of DREB /CBF to provide reference for plant stress?resistant breeding.DREB /CBF transcription factor,

with a typical AP2/EREBP DNA?binding domain,could specifically bind to the DRE /CRT (Dehydration Responsive Element /C?repeat)cis?acting element and activate a lot of the expression of stress inducible genes under dehydration,low temperature and saline conditions,and hence increase plants’tolerance to environmental stresses.According to the published literature in last decades,a vast number of DREB /CBF transcription factor genes have been isolated and

characterized from a variety of plants such as Arabidopsis thaliana ,Brassica napus ,Oryza sativa ,Zea mays ,Triticum

aestivum ,Gossypium hirsutum ,Glycine max and Lycopersicon esculentum .Over?expression of these DREB /CBF

cDNAs in Arabidopsis thaliana ,Brassica napus ,Lycopersicon esculentum ,Triticum aestivum and Populus could greatly enhance stress tolerance of these transgenic plants,which indicated the importance of DREB /CBF transcription factors in plant stress?resistant breeding.

Key words :　DREB/CBF;transcription factor;DRE/CRT cis?acting element;transgenic plant;stress?resistant breeding 干旱、盐碱、低温等非生物逆境是影响植物生长

发育的主要因素,植物受到逆境胁迫时会产生形态、生理、基因表达等适应性调节反应以降低或消除危

害。转录因子(反式作用因子)基因是植物中最重要的一类调节基因,其在植物体内构成复杂的调节网络,在时间和空间上协同控制基因的表达。转录

　第1期李科友等:植物非生物逆境胁迫DREB/CBF转录因子的研究进展

因子是能够与真核基因启动子区域中顺式作用元件发生特异性作用的DNA结合蛋白,通过它们之间以及与其他相关蛋白之间的相互作用,激活或抑制基因转录。DREB/CBF(Dehydration Responsive

Element Binding Protein/C?repeat Binding Factor)转录因子,即干旱应答元件结合蛋白质/C-重复序列结合子,能特异结合DRE/CRT(Dehydration Responsive Element/C?repeat)顺式作用元件。DRE/CRT顺式作用元件普遍存在于干旱、高盐或低温等逆境应答基因的启动子中,核心序列为CCGAC。DREB/CBF转录因子由逆境胁迫诱导产生后,可激活其他一系列依赖DRE/CRT顺式作用元件的抗逆功能基因的表达,从而增强植物对干旱、低温及高盐等逆境的抗性(Agarwal et al.,2006)。因此,DREB/CBF转录因子在植物抗逆中的作用越来越广泛地受到重视,目前,已成为植物抗逆分子生物学研究的热点之一。本文就近十几年特别是近5年来国内外DREB/CBF转录因子的研究进展, DREB/CBF转录因子在植物抗逆中的作用及研究作一综述,旨在为植物抗逆育种提供理论依据。

1　DREB/CBF转录因子的结构特点

迄今报道的DREB/CBF转录因子基因内均无内含子,从蛋白质结构分析,DREB/CBF转录因子含有1个保守的AP2/EREBP结构域,是转录因子AP2/EREBP家族中的1个亚家族,主要调节植物对低温、干旱和高盐等逆境的分子应答。DREB/CBF 亚族又分为6个亚组(A?1—A?6),氨基酸序列对比发现,A?1类转录因子上游含有1个保守的核定位信号区(NLS):PKRPAGRTKFRETRHP,下游含有DSAW的保守序列和羧基末端的LWSY保守序列,这些序列被称为A?1类转录因子的特征序列(Jaglo et al.,2001)。而在与干旱、高盐诱导相关的A?2类转录因子中不存在(Yamaguchi et al.,2006)。A?3—A?64个亚组占整个拟南芥(Arabidopsis thaliana) DREB/CBF亚族基因的75%,但目前对它们的功能研究很少。最近,对这4个DREB/CBF亚组的基因研究相继有一些报道,如拟南芥TINY2(A?4)(Wei et al.,2005),大豆(Glycine max)Gm?DREB2(A?5) (Chen et al.,2007),陆地棉(Gossypium hirsutum) GhDBP3(A?4),GhDBP1(A?5),GhDBP2(A?6) (Huang et al.,2006a)和玉米(Zea mays)ZmDBF1 (A?6)(Kizis et al.,2002)。

DREB/CBF转录因子的二级结构具有典型的结构特征:在C-末端富含酸性氨基酸,只有少量碱性氨基酸,功能是作为转录激活区。例如小麦(Triticum aestivum)的TaDREB1C-末端的50个氨基酸中酸性氨基酸占25%,碱性氨基酸仅为4%;榆钱菠菜(Atriplex hortensis)的AhDREB1C-末端的80个氨基酸中酸性氨基酸占23%,碱性氨基酸仅为3%(Shen et al.,2003a;2003b)。N-末端富含碱性氨基酸,是核定位信号区;中间由58个左右氨基酸残基组成的AP2/EREBP结构域,分为2个保守元件YRG和RAYD。YRG含有19~22个氨基酸残基,大多数为碱性氨基酸残基,有利于与DNA的结合。RAYD由42或43个氨基酸残基组成,含有由18个氨基酸组成的α-双亲螺旋的核心区域; AP2/EREBP结构域可形成3个β-折叠和1个α-螺旋结构,其中位于第2个β-折叠中的第14位的缬氨酸(V)和第19位的谷氨酸(E),特别是第14位的缬氨酸(V),对决定DREB/CBF转录因子与DRE/CRT顺式作用元件的特异性结合起关键作用(Liu et al.,1998;Sakuma et al.,2002)。已报道的DREB/CBF转录因子的AP2/EREBP结构域中有7个关键残基参与了DREB/CBF蛋白与DRE/CRT顺式作用元件的直接作用,它们是4个精氨酸(R)残基、2个色氨酸(W)残基和1个缬氨酸(V)残基(陈金焕等,2007)。Gao等(2002)通过突变的方法发现缬氨酸(V)的突变导致DREB/CBF蛋白结合DRE/ CRT顺式作用元件的活性基本丧失,从而说明缬氨酸(V)是DREB/CBF家族的特征残基。

2　DREB/CBF转录因子基因的克隆

自Stockinger等(1997)首次从拟南芥cDNA文库中克隆到AtCBF1以来,DREB/CBF转录因子基因的克隆得到较大的发展,已陆续从各种植物中克隆出DREB/CBF基因(表1)。

3　DREB/CBF转录因子基因的表达调控Stockinger等(1997)利用酵母一元杂交方法首次从拟南芥cDNA文库中克隆到DRE/CRT结合蛋白质基因,并命名为CBF1(CRT-binding factor 1)。Liu等(1998)利用rd29A基因启动子的DRE/ CRT顺式作用元件和酵母一元杂交方法,从拟南芥中克隆了2类共5个与DRE元件特异结合,在低温、干旱或高盐胁迫下调控基因表达的DREB转录因子,分别命名为DREB1A(CBF3),DREB1B (CBF1),DREB1C(CBF2)和DREB2A,DREB2B。自那时以来,DREB/CBF转录因子一直是植物分子生物学研究的热点。

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表1　从不同种植物中分离的DREB /CBF 基因对各种非生物胁迫的反应①

Tab.1　DREB /CBF genes isolated from different plants and their transcript respones to various abiotic stress

物种Species

DREB /CBF 类别DREB /CBF type 胁迫诱导表达条件Expression in stress

低温Cold

干旱Drought

盐Salt ABA 参考文献References

拟南芥Arabidopsis thaliana

AtDREB1A /AtCBF3AtDREB1B /AtCBF1AtDREB1C /AtCBF2AtDREB1D /AtCBF4+++-

---+

---+---+Liu et al.,1998Gilmour et al.,1998Medina et al.,1999Haake et al.,2002Sakuma et al.,2002AtDREB2A AtDREB2B AtDREB2C AtDREB2D

----++--++++----Liu et al.,1998Sakuma et al.,2002AtDREB1E /DDF2AtDREB1F /DDF1AtDREB2E ------++---+Magome et al.,2004Sakuma et al.,2002水稻Oryza sativa

OsDREB1A OsDREB1B OsDREB1C OsDREB1D OsDREB2A OsDREBL OsDREB1?1OsDREB4?1OsDREB4?2OsDREB1F ++??--+??-??+--??-+-??+??++-??-+-??+??+--??---??-??+Dubouzet et al.,2003

Chen et al.,2003Dubouzet et al.,2003Wang et al.,2008欧洲油菜Brassica napus

BnCBF5BnCBF7BnCBF16BnCBF17++++----???--?-?Gao et al.,2002

大豆Glycine max

GmDREBa GmDREBb GmDREBc GmDREB2GmDREB5++-+-+++++++++++-++-Li et al.,2005Chen et al.,2007于月华等,2008小麦Triticum aestivum

TaDREB6CBF2?1CBF2?2WDREB2?+++++++???+---+倪志勇等,2008Kume et al.,2005Egawa et al.,2006小偃54Triticum aestivum ‘Xiaoyan 54’TaDREB1++++Shen et al.,2003a 大麦Hordeum vulgare HvCBF3HvDRF1+-?-Choi et al.,2002Xue et al.,2004陆地棉Gossypium hirsutum

GhDREB1L GhDBP2GhDBP3+++

+++

+++

?++

Huang et al.,2007Huang et al.,2006a Huang et al.,2006b 番茄Lycopersicon esculentum CBFlike +???Jaglo et al.,2001烟草Nicotiana tabacum Tsil ??+?

Park et al .,2001落花生Arachis hypogaea PNDREB1++--张梅等,2009苇状羊茅Festuca arundinacea FaDREB1+---阳文龙等,2006黑麦草Lolium perenne LpCBF3+---Xiong et al.,2006辣椒Capsicum annuum CaDREBLP1-++-Hong et al.,2005番薯Ipomoea batatas swDREB1+++-

Kim et al .,2008榆钱菠菜Atriplex hortensis AhDREB1??+?Shen et al .,2003b 狗牙根Cynodon dactylon BeDREB1BeDREB2++

??++??谢永丽等,2005狼尾草Pennisetum glaucum PgDREB 2A +++?

Agarwal et al .,2007玉米Zea mays

ZmDREB2A ZmDBP2ZmDBF1+??-++

-++

-++

Qin et al .,2007Wang et al.,2010Kizis et al.,2002盐芥Thellungiella salsuginea TsCBF1????高峰等,2005巴西橡胶树Hevea brasiliensis

HbCBF1

?

?

?

?

程汉等,2005

6

21

　第1期

李科友等:植物非生物逆境胁迫DREB /CBF 转录因子的研究进展

续表Continued

物种Species

REB /CBF 类别DREB /CBF type

胁迫诱导表达条件Expression in stress

低温Cold

干旱Drought

盐Salt ABA ABA

参考文献References

蓝桉Eucalyptus globulus EgCBF1+???Gamboa et al .,2007芦荟Aloe vera var.chinensis AvDREB1AvDREB2++

?+?+?+Wang et al .,2007张倩等,2009银新杨Populus alba ×P.alba var.pyramidalis

PaDREB 2+++?

秦红霞等,2005河北杨Populus hopeiensis PhCBF4a PhCBF4b ??++????Wang et al.,2008小立碗藓Physcomitrella patens PpDBF1++++Liu et al .,2007毛华菊Dendranthema vestitum DvDREB2++++Liu et al .,2008菊花Dendranthema morifolium DmDREBa DmDREBb ++????++Yang et al .,2009西府海棠Malus micromalus MrDREBA6++++付晓燕等,2009盐角草Salicornia brachiata SbDREB2A ?++?Gupta et al .,2010柠条锦鸡儿Caragana korshinskii CkDBF ++++Wang et al .,2010b 枳Poncirus trifoliata PtCBFb +???Wang et al .,2009越橘Vaccinium vitis?idaea VviDREB1+?++Wang et al .,2010c 截形苜蓿Medicago truncatula MtDREB1C +???Chen et al .,2010毛白杨Populus tomentosa

PtCBF5

+

+

-

-

周洲等,2010

①+:诱导表达Induction expression ;-:诱导不表达Induction not expression;?:未研究Not studied;??:组成型表达Constitutive expression.

植物的干旱、高盐及低温胁迫应答途径涉及到

依赖于ABA 和不依赖ABA 的信号传导途径。目前,从植物中分离得到的DREB /CBF 类转录因子都

能通过与DRE /CRT 元件的相互作用,完成对干旱、高盐或低温胁迫应答基因的激活作用。其中A?1和A?2组的主要成员DREB1A /B /C 和DREB2转录因子分别参与到不依赖于ABA 的低温和脱水(干旱或

高盐)胁迫应答途径中,其他受逆境诱导的DREB /CBF 转录因子如DREB1D /CBF4,GmDREBa,GmDREB2,ZmDBP2,ZmDBF1,CkDBF,MrDREBA6,TaDREBA6,PpDBF1,DvDREB,AvDREB2,GhDBP2

和GhDBP3等参与到依赖ABA 的脱水应答途径中。这些结果表明不同植物中类似的DREB /CBF 基因,其参与的细胞内信号转导的途径可能不同,植物在逆境胁迫应答反应中存在着复杂的信号传递途径,并且各种胁迫信号传递途径间通过某些共同的组分联系在一起,构成一个复杂的信号传递网络(Haake et al.,2002)。大部分DREB /CBF 基因的表达受干旱、盐碱、低温或冻害等逆境胁迫的诱导,但不同形式DREB /CBF 在同一植物和不同植物中诱导表达的条件和所需时间不同。

在正常生长条件下,在拟南芥中几乎检测不到AtDREB1基因转录产物的存在;当受到低温协迫时,AtDREB1A ,AtDREB1B 和AtDREB1C 基因能在

15min 内表达,并在2h 内达到最高,随后开始下降,但在24h 内基因表达仍然高于对照;而对于AtDREB2基因来说,干旱和高盐处理时,10h 表达

水平达到最高(Liu et al .,1998)。AtCBF4基因被干

旱和ABA 处理快速强烈诱导,而不受低温诱导(Haake et al .,2002)。水稻低温处理后40min 内

OsDREB1A 和OsDREB2A 基因被快速诱导,但不受外源ABA 的诱导。OsDREB1A 盐处理5h 后被诱

导,OsDREB1C 为组成型表达,OsDREB1D 在胁迫和正常条件下检测不到,OsDREB2A 干旱或高盐250mmol ·L -1NaCl 处理24h 被诱导,对低温和ABA 处理反应不敏感(Dubouzet et al.,2003)。辣椒(Capsium annuum )中CaDREBLP1被干旱、高盐快速诱导,机械损伤也可诱导,但不被低温诱导,这种表达方式与AtDREB2A 表达方式十分相似,但还属于DREB1类基因,因为其结构特征与其他DREB1基因类似,这说明CaDREBLP1属于一种新型的DREB 基因(Hong et al.,2005)。

DREB /CBF 转录因子家族可以特异性地识别并结合DRE /CRT 顺式作用元件,调控RD29A ,RD17,ERD10,KIN1,COR6.6,KIN2和COR15A 等多种下游基因(启动子区域含有DRE /CRT 顺式作用元件)的表达,其自身的表达也受上游基因作用、“同伴”作用以及其下游基因的反馈作用。研究表明,DREB /CBF 的上游基因ICE1(inducer of CBF expression 1),LOS1(low expression of osmotically respessive genes 1),LOS4(low expression of osmotically respessive genes 4),FRY2(fiery2),HOS1(high expression of osmotically respessive genes 1)等

对DREB /CBF 的表达都起着重要的调控作用(Chinnusamy et al.,2003;Lee et al.,2001)。Novillo 等(2004)通过基因芯片技术发现,在低7

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林业科学47卷　

温情况下,AtDREB1A /AtCBF3和AtDREB1B /AtCBF1较AtDREB1C /AtCBF2提前表达。此外,在拟南芥cbf2突变株中,AtDREB1A /AtCBF3和AtDREB1B /AtCBF1的表达量都有所增加,其抗旱耐盐抗冻的能力也较野生型强。这些研究说明AtDREB1C /AtCBF2

对

AtDREB1A /AtCBF3

和

AtDREB1B /

AtCBF1起到了反向调节的作用(Novillo et al .,

2004)。Gao 等(2002)发现在欧洲油菜(Brassica

napus )los1突变株中,由低温诱导的含有DRE /CRT 元件的基因合成受阻时CBF 的表达量却增高,推测CBF /DREB1可能被自己的基因产物和下游基因产物所反馈抑制。

有关特定组织中DREB /CBF 表达的研究还不多。在正常生长条件下,AtDREB2A 和AhDREB1在拟南芥和山波菜根、茎和叶中均得到表达,在盐胁迫下,AhDREB1在根中高度表达,但在茎叶中表达量差异不大(Shen et al .,2003b)。在大豆幼叶中GmDREBa

被低温、干旱、高盐快速诱导,而GmDREBb 的表达则不明显,但干旱、盐和ABA 处理后GmDREBc 在根中表达程度很高(Li et al .,2005)。菊花(Dendranthema morifolium )DmDREBa 和DmDREBb 在茎和叶中的表达量比在根和花中的表达量高(Liu et al .,2008)。番薯(Ipomoea batatas )SwDREB1在茎和块状根中高水平表达(Kim et al .,2008)。西府海棠(Malus micromalus )MrDREB6在叶中的表达量远高于其他组织,其次在种子中,在茎中的表达量最少(付晓燕等,

2009)。这种组织特异性表达可能与植物的不同抗性机制有关,受启动子特异元件调控。

植物细胞从感应干旱、高盐或低温胁迫开始,通过各级信号传递DREB /CBF 转录因子与DRE /CRT 顺式作用元件相互作用的转录调控,目的基因的表达、基因产物的积累引起的各种生理生化调节,最终使植物抗逆性获得增强,这一系列过程可用图1来概括(刘强等

,2000)。

图1　逆境胁迫下DREB /CBF 转录因子的感应和表达调控过程

Fig.1　A process of induction,expression and regulation of DREB /CBF transcription factor in environmental stresses

4　DREB /CBF 转录因子基因在植物抗逆基因工程中的作用

由于植物对低温、干旱和高盐等逆境胁迫耐性受多个基因的控制,因此其强弱往往不取决于单个

因子,而是受许多因子影响。DREB /CBF 转录因子能够识别DRE /CRT 元件,与基因启动子区域的DRE /CRT 顺式作用元件特异性结合,在信号传导及诱导下游许多抗逆相关的功能基因表达中起到关键作用。因此,利用转录因子增强植物抗逆性成为改

8

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　第1期李科友等:植物非生物逆境胁迫DREB/CBF转录因子的研究进展

良植物抗逆性的重要途径。DREB/CBF转录因子自首次报道以来,一直受到广泛关注,研究者们通过转基因手段鉴定DREB/CBF转录因子的功能,同时也得到了许多抗性增强的转基因植株(表2)。

表2　过量表达DREB/CBF的转基因植物对逆境胁迫的反应

Tab.2　Stress response of transgenic plants overexpressing DREB/CBF

目的基因

Purpose gene转基因植物

Transgenic plants转基因植株的抗性

Tolerance of transgenic plants参考文献

References AtCBF1拟南芥Arabidopsis thaliana低温Freezing Joglo et al.,1998 AtDREB1A拟南芥Arabidopsis thaliana低温、干旱Freezing and drought Liu et al.,1998 AtDREB1A拟南芥Arabidopsis thaliana低温、干旱、高盐Freezing,drought and high?salt Kasuga et al.,1999 AtDREB1A地被菊Dendranthema grandiflorum干旱、高盐Drought and high?salt洪波等,2006 AtDREB2A CA拟南芥Arabidopsis thaliana干旱Drought Sakuma et al.,2006 AtCBF3拟南芥Arabidopsis thaliana低温Freezing Gilmour et al.,2000

AtCBF1欧洲油菜Brassica napus低温Freezing甄伟等,2000

Joglo et al.,2001 AtCBF1烟草Nicotiana tabacum低温Freezing甄伟等,2000 AtCBF2欧洲油菜Brassica napus低温、干旱Freezing and drought Joglo et al.,2001

AtCBF1番茄Lycopersicon esculentum低温、干旱和氧化胁迫

Freezing,drought and oxidative stress Hsieh et al.,2002a;2002b Lee et al.,2003

AtCBF1草莓Fragaria×ananassa低温Freezing Owens et al.,2002 AtCBF4拟南芥Arabidopsis thaliana低温、干旱Freezing and drought Haake et al.,2002 AtDREB1A烟草Nicotiana tabacum低温、干旱Freezing and drought Kasuga et al.,2004 AtDREB1A小麦Triticum aestivum干旱Drought Pellegrineschi et al.,2004 AtCBF1黑麦草Lolium perenne干旱Drought杨风萍等,2006 AtCBF1杨树Populus低温Freezing Benedict et al.,2006

AtCBF1水稻Oryza sativa低温、高盐、渗透胁迫

Freezing,high?salt and osmotic stress Lee et al.,2004 Oh et al.,2005 Ito et al.,2006吴关庭等,2006

AtDREB1A苇状羊茅Festuca arundinacea干旱Drought Zhao et al.,2007 AtDREB1A落花生Arachis hypogaea干旱Drought Bhatnagar?Mathur et al.,2007 AtDREB1A马铃薯Solanum tuberosum低温、高盐Freezing and high?salt Behnam et al.,2007;2006

Celebi?Toprak et al.,2005

AtDREB1c南林895杨Populus×

euramericana‘Nanlin895’干旱、高盐Drought and high?salt杨春霞等,2009 AhDREB1烟草Nicotiana tabacum干旱、高盐Drought and high?salt Shen et al.,2003b OsDREB1A拟南芥Arabidopsis thaliana低温、干旱、高盐Freezing,drought and high?salt Dubouzet et al.,2003 BNCBF5欧洲油菜Brassica napus低温Freezing Savitch et al.,2005 BNCBF17欧洲油菜Brassica napus低温Freezing Savitch et al.,2005

Tsil1烟草Nicotiana tabacum渗透胁迫Osmotic stress Park et al.,2001 ZmDREB1/CBF拟南芥Arabidopsis thaliana低温、干旱Freezing and drought Qin et al.,2004 GmDREB2小麦Triticum aestivum干旱、高盐Drought and high?salt高世庆等,2005 GmDREB3拟南芥Arabidopsis thaliana低温、干旱、高盐Freezing,,drought and high?salt Chen et al.,2009 GmDREB2拟南芥Arabidopsis thaliana干旱、高盐Drought and high?salt Chen et al.,2007 HsDREB1A百喜草Paspalum notatum干旱、高盐Drought and high?salt James et al,.2008 LpCBF3拟南芥Arabidopsis thaliana低温Freezing Zhao et al.,2008 WDREB2烟草Nicotiana tabacum低温、渗透胁迫Freezing and osmotic stress Kobayashi et al.,2008 GhDREB1烟草Nicotiana tabacum低温Freezing Shan et al.,2007 GmDREB1紫苜蓿Medicago sativa高盐High?salt Jin et al.,2010 GmDREB5烟草Nicotiana tabacum干旱、高盐Drought and high?salt于月华等,2008 TsCBF1玉米Zea mays干旱Drought Zhang et al.,2010 SbDREB2A大肠杆菌Escherichia coli BL21高盐High?salt Gupta et al.,2010 PgDREB2A烟草Nicotiana tabacum高离子、高渗透胁迫Hyperionic and hyperosmotic stresses Agarwal et al.,2010 CkDBF烟草Nicotiana tabacum高盐、渗透胁迫High?salt and osmotic stress Wang et al.,2010 MtDREB1C截形苜蓿Medicago truncatula低温Freezing Chen et al.,2010 MtDREB1C月季花Rosa chinensis低温Freezing Chen et al.,2010 ZmDBP2拟南芥Arabidopsis thaliana干旱Drought Wang et al.,2010

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DREB/CBF转录因子基因在转基因植物中表达较为复杂。目前,在植物抗逆基因工程中主要利用组成性启动子和特异诱导性启动子构建DREB/ CBF转基因抗逆品系。

利用组成型CaMV35S启动子在拟南芥中过量表达的AtDREB1A,OsDREB1A或GmDREB2(Kasuga et al.,1999;Dubouzet et al.,2003;Chen et al.,2007),在水稻中过量表达的AtDREB1A(Oh et al.,2005)和在烟草中过量表达的AhDREB1,GhDREB1(Shen et al.,2003a;Shan et al.,2007)均能增强植物抗逆性,但在一些情况下过量表达的DREB/CBF导致植株生长矮化(Liu et al.,1998;Kasuga et al.,1999; Dubouzet et al.,2003)。这可能是由于组成型CaMV35S启动子的控制,外源基因在转基因植物所有发育阶段都会持续表达,造成植物体内资源的非必要浪费,此外大量外源蛋白的积累也会干扰植物正常的代谢调控;使用特异诱导性rd29A启动子可以消除矮化现象(Kasuga et al.,1999;Pellegrineschi et al.,2004;Sakuma et al.,2006)说明诱导型启动子rd29A能更有效地提高植物对低温、干旱和高盐的耐受性(Kasuga et al.,1999;Agarwal et al.,2006),因为诱导型启动子是在遇到胁迫信号时才启动基因的表达,不会影响植物体在正常情况下的生长发育。Hsieh等(2002a)将与CaMV35S启动子连接的AtCBF1基因转入番茄(Lycopersicon esculentum),发现转基因番茄体内有类似拟南芥中的DREB信号转导过程,同时发现自由基减少,脯氨酸含量升高,植株的抗旱性和耐低温性都明显增强,但是也出现生长矮化现象;用GA3处理后,植株恢复正常生长,且耐寒能力仍明显高于野生型植株。但是CaMV35S 启动子驱动AtDREB1A/AtCBF3过量表达的转基因番茄和拟南芥,用GA3处理后,矮化现象并不发生变化(Hsieh et al.,2002a;2002b)。因此,转基因植株产生的矮化现象可能并非GA生物合成干扰,而是其他机制所致。

将AtDREB2A基因转入到拟南芥中,过度表达AtDREB2A的转基因拟南芥没有增强对逆境的抗性,由此推测AtDREB2A基因产物激活下游靶基因可能需要磷酸化或去磷酸化修饰(Liu et al.,1998; Dubouzet et al.,2003)。Sakuma等(2006)研究发现在拟南芥AtDREB2A基因中第136~165位氨基酸残基抑制了DREB2A蛋白质的活性,将该区域删除后,过度表达DREB2A的转基因拟南芥提高了对干旱的抗性。Agarwal等(2007)研究发现狼尾草(Pennisetum glaucum)PgDREB2A是一个磷酸化蛋白,并且磷酸化作用负调控蛋白对DNA的结合活性,磷酸化后的蛋白不能结合DRE元件,当用磷酸酶处理后,PgDREB2A结合DRE元件的能力得到了恢复。但是,Dubouzet等(2003)在另一个试验中,分别将拟南芥和水稻的OsDREB2A基因转入到水稻原生质体中却激活了GUS基因的表达。所以, DREB2A基因产物激活下游靶基因需要磷酸化或去磷酸化,这一结论并不是转基因植株胁迫抗性没有提高的真正原因,探明DREB2A基因如何提高植物的抗性还需要做进一步的研究。

Chen等(2007)从大豆(Glycine max)中克隆得到受干旱、高盐、低温以及ABA诱导的转录因子GmDREB2基因,将其分别置于特异启动子rd29A和CaMV35S启动子下转入拟南芥后,2种转基因植株的生长情况和野生型植株相比均未表现出异常,逆境处理试验表明转基因植株的抗性明显提高。目前,已从大豆中分离出6个DREB同源基因GmDREBa,GmDREBb,GmDREBc,GmDREB1, GmDREB2和GmDREB3(Li et al.,2005;Chen et al., 2007;2009),其中GmDREB1,GmDREB2和GmDREB3同属于DREB家族的A?5亚家族,过量表达的GmDREB1,GmDREB2和GmDREB3可以增强转基因拟南芥植株的抗旱、抗低温和抗高盐性(Chen et al.,2007;2009;Jin et al.,2010),这表明DREB家族的亚家族A?5是提高植物抗逆性的重要基因资源。

总之,一系列的转基因研究结果表明,DREB/ CBF转录因子家族不论是在双子叶植物、单子叶植物,还是在草本植物及木本植物抗逆品种改良中均具有十分重要的应用价值。

5　DREB/CBF转录因子研究中的问题与展望

综上所述,DREB/CBF在植物逆境信号转导途径中具有重要的位置,已对DREB/CBF的结构、类型、表达特性,在逆境胁迫下的作用及在培育转基因抗逆植物方面进行了大量的研究工作,这对于深刻理解植物的抗逆机制及利用该机制进行抗逆新品种的培育有重要意义,但依然存在许多问题有待进一步研究。

第一,DREB/CBF对于上游调控基因的研究还有待加强。Chinnvsamy等(2003)在拟南芥中发现

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　第1期李科友等:植物非生物逆境胁迫DREB/CBF转录因子的研究进展

了一个转录因子ICE1,该基因编码1个MYC?type bHLH(basic helix?loop?helix)蛋白,能够调节DREB1A/CBF3基因的表达,但是对其他DREB1/ CBF基因没有明显的调节作用。冷信号激活ICE1表达的机制,ICE1在冷信号和DREB1/CBF之间的具体调节过程,细胞膜上特异冷信号受体的分离鉴定,理清包括Ca2+作为第2信使瞬时积累到ICE1的转录后修饰之间的上游事件等,还有待以后研究。

第二,有关DREB/CBF类抗逆境转录因子的功能及转录调控的研究大部分来自拟南芥,而对其他植物的研究相对较少。Benedict等(2006)发现杨树和拟南芥中的调节子(regulon)相似,但是对这些调节子的上游序列进行分析,并没有发现DRE (CCGAC)的大量存在,而ABRE却比较丰富,这2个顺式元件在杨树调节子中的相互作用关系也有待继续研究。已完成的杨树基因组测序为木本植物DREB/CBF调控途径的研究提供了有力的条件,对DREB/CBF在木本植物中的调控网络进行解析,并将其应用于优质抗逆林木的筛选,将是今后研究的一个重要方向。

第三,已对DREB/CBF类抗逆境转录因子在干旱、低温和高盐胁迫的应答进行了大量的研究,而对它们在其他胁迫如高温、高湿和抗氧化等胁迫方面的研究较少;DREB/CBF转录因子识别各种不同顺式作用元件并与之结合的机制,DREB/CBF转录因子影响转录起始速度的机制和如何接受外源信号以及如何控制靶基因的表达还不够清晰。

第四,转入DREB/CBF基因受体植物种类不够广泛,尚缺乏能够在田间应用的抗逆转基因植物。例如转CBF基因时,所研究的对象主要集中在拟南芥、欧洲油菜、番茄、大麦、小麦等植物上,对于一些亲缘关系较远的植物是否有效,还有待进一步研究;有效调控外源DREB/CBF基因表达,目前除了rd29A基因启动子控制DREB/CBF基因外,缺乏更多的探索。

DREB/CBF转录因子所介导的基因表达调控网络在植物抵御各种环境胁迫的应答中具有重要功能,在感知胁迫信号到胁迫应答基因表达的整个信号传导网络中,各种转录因子和顺式元件间的相互作用构成了基因表达调控的基础。植物对胁迫应答的反应是由多基因协同控制的。利用生物技术改良农林作物的抗逆性,针对性强,效率高。在抗逆分子育种中,与导入或改良个别功能基因来提高某种抗性的方法相比,导入或改良一个转录因子是提高植物抗逆性更为有效的方法和途径。鉴于DREB/CBF 转录因子在植物抗逆中的特殊地位,相信在不远的将来,随着对其在植物抗逆中分子机制的深入研究,

DREB/CBF转录因子在培育抗逆植物新品种方面会取得新的重要进展,将有巨大的应用前景。

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