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Research Progress in Gene Cloning in Forest Trees

林木基因克隆研究进展



全 文 :植物学报 Chinese Bulletin of Botany 2011, 46 (1): 79–107, www.chinbullbotany.com
doi: 10.3724/SP.J.1259.2011.00079

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收稿日期: 2010-06-22; 接受日期: 2010-11-08
基金项目: 转基因生物新品种培育科技重大专项(No.2009ZX08009-098B)和 973计划(No.2009CB119107)
* 通讯作者。E-mail: suxh@caf.ac.cn
林木基因克隆研究进展
李少锋, 苏晓华*, 张冰玉
中国林业科学研究院林业研究所, 国家林业局林木培育重点实验室, 北京 100091
摘要 林木种质资源丰富, 种质间遗传差异大, 控制林木重要性状的基因克隆及转化对培育优良林木新品种具有很强的实
用价值, 但许多具有潜在应用价值的林木基因未得到充分发掘和有效分离。近年来, 随着各种不同林木cDNA文库的建立,
大规模随机EST测序技术的运用以及克隆技术的不断完善, 特别是毛果杨(Populus trichocarpa)基因组测序计划的完成,
大量与林木重要性状相关的基因被分离和鉴定。这些重要基因的获得为利用转基因技术培育高产、优质、抗逆、抗病虫害
的林木新品种奠定了一定的基础。该文综述了20多年来国内外林木基因克隆的研究进展, 对基因克隆及其应用过程中亟待
解决的问题进行了讨论, 并对其发展趋势进行展望。
关键词 林木, 基因克隆, 研究进展, 转基因技术
李少锋, 苏晓华, 张冰玉 (2011). 林木基因克隆研究进展. 植物学报 46, 79–107.
林木在净化大气、防风固沙、保持水土、维持生
态平衡和生物多样性、提供优质木材和高产优质林果
等方面发挥着突出作用, 具有巨大的生态效益、社会
效益和经济价值。传统的育种手段如引种驯化、杂交
育种、选择育种等难以实现林木高抗优质多性状综合
改良的需求。近年来生物技术的发展、分子标记辅助
选择技术的运用以及基因工程研究的兴起, 为利用基
因工程手段改良林木、加速林木新品种的选育奠定了
基础。林木等木本植物基因资源丰富, 遗传多样性复
杂, 但许多天然优良基因尚未被分离利用, 大部分基
因的功能仍处于未知状态。各种模式植物基因组测序
的完成, 特别是杨树基因组测序计划的完成, 并结合
连锁和连锁不平衡的分析方法, 大大促进了木本植物
功能基因的克隆和功能基因组学的研究 (尹佟明 ,
2010)。
近年来 , 林木功能基因陆续被分离和鉴定。
Yamamoto等(1988a)从黑松(Pinus thunbergii)中克
隆了首个来自林木的基因——核酮糖二磷酸羧化酶
基因。到目前为止, 为达到改良材性、缩短花期、抗
干旱和病虫害等育种目标, 科研人员分离的林木来源
的基因总数达到470个以上, 运用到的克隆技术主要
有: RACE技术、RT-PCR技术、mRNA差别显示技术
(DDRT-PCR)、抑制性差减杂交(suppression sub-
tractive hybridization, SSH)、核酸探针分离目标基因
和酵母单杂交技术等。对这些基因的分析鉴定, 主要
采用与拟南芥(Arabidopsis thaliana)、烟草(Nicoti-
ana tabacum)等的同源基因进行相似性比较, 构建
分子进化树, 或对蛋白质结构域和功能进行预测分
析; 通过半定量和定量RT-PCR技术, 研究目的基因
在某种时空状态或胁迫环境下的表达量。运用反向遗
传学方法, 通过基因超量表达技术、反义RNA技术、
RNA干涉和转基因技术研究林木基因的功能, 填补
或完善基因功能注释, 阐述多年生林木生物学性状的
基因表达调控机制正成为当前林木基因克隆及应用
研究的热点。本文综述了近20年来国内外林木基因克
隆的研究进展, 并对今后该领域的发展趋势进行了
展望。
1 国外林木基因克隆研究概况
近几年国外在林木的木材形成调控和花发育机制、抗
旱耐盐和抗氧化胁迫等方面做了大量研究, 从杨树、
松树、桉树、柳树、柽柳(Tamarix chinensis)、白桦
(Betula platyphylla)和银杏(Ginkgo biloba)等植物中
·专题论坛·
80 植物学报 46(1) 2011
克隆获得了387个功能基因, 其中参与木材形成的有
87个, 花发育49个, 抗冻21个, 抗旱9个, 抗病9个,
抗虫3个, 防御机械损伤3个, 抗氧化7个, 耐重金属
污染5个及其它功能的基因194个。上述各基因及其功
能详见表1和表2。
1.1 与材质有关的基因
木质素生物合成途径中的关键酶基因PtCOMT、F5H、
4CL、CCR、CCoAOMT和CAD等均已从毛果杨
(Populus trichocarpa) 、 美 洲 山 杨 (Populus
tremuloides) 、 火 炬 松 (Pinus taeda) 中 克 隆
(Doorsselaere et al., 1995; Hu et al., 1998; Li et al.,
1999, 2005; Sibout et al., 2002; Bhuiya and Liu,
2009)。利用基因工程技术对这些基因进行表达调控,
可以有效改变木质素组成或降低木质素含量, 培育新
型能源品种, 从源头降低造纸成本, 减少环境污染
(高原等, 2007)。除此以外, Ko等(2005)从欧洲山杨×
毛 白 杨 杂 交 杨 (Populus tremula × Populus
tomentosa)中克隆到 class III HD-Zip转录因子
PtaHB1基因, 该基因被证实在维管组织分化中起调
控作用。Samuga和Joshi(2004)从美洲山杨中分离到
PtrCSLD2全长基因, 其在木质部次级细胞壁有较高
丰度的表达 , 参与纤维素生物合成。PoptrMP1和
PoptrMP2属于MONOPTEROS(MP)/AUXIN响应因
子, PoptrMP1集中在毛果杨次生木质部表达, 过量
表达PoptrMP1导致作用的下游靶基因的转录量提高
2–4倍 , 两者可能在维管组织的发育中起作用
(Johnson and Douglas, 2007)。Sonoda等(2009)将
赤桉(Eucalyptus camald- ulensis)HD-Zip class II转
录因子EcHB1基因转入烟草, 转基因植株纤维长度
和干重增加, 叶片、根部和茎部的生长量均明显高于
对照。Lee等(2009)的研究证明, RNA干扰(RNAi)技
术抑制PoGT47C的表达后, 转基因银白杨×欧洲山
杨杂交杨(Populus alba × Populus tremula)植株次
生细胞壁厚度明显减小, 葡糖醛酸木聚糖的合成受
阻, 导管外观变形, 纤维素含量下降。最近, MYB转
录因子PtrMYB3和 Ptr- MYB20及NAC转录因子
PtrWND2B和PtrWND6B均被证实参与纤维素和木
质素的生物合成, 并参与调控木材形成(McCarthy et
al., 2010; Zhong et al., 2010)。
1.2 与生殖有关的基因
林木树种存在生长周期长、生殖发育滞后的特点。目
前已从美洲山杨、辐射松 (Pinus radiata)和巨桉
(Eucalyptus grandis)中分离了一些参与花形成和发
育的基因——MYB、PTM、PMADS2、PTLF、PRFLL、
NEEDLY、FT2, 为通过基因工程手段进行开花调控,
缩短花周期, 获得提早开花或花期不育的转基因新品
种打下良好基础(Mellerowicz et al., 1998; Mouradov
et al., 1998; Rottmann et al., 2000; Cseke et al.,
2003a, 2003b; Liu et al., 2003; Hsu et al., 2006;
Mellway et al., 2009)。花发育基因PTM3/4为美洲山
杨SEPALLATA-class MADS-box基因, 其在转基因
烟草、拟南芥、美洲山杨中均参与季节性的开花调节
(Cseke et al., 2005)。辐射松的NEEDLY (NLY)基因
与拟南芥LEAFY/FLORICAULA基因高度同源, 转基
因LFY::NLY植株可以互补花发育途径中对应基因
LFY的缺失 , 表明NLY与LFY在功能上具有相似性
(Mouradov et al., 1998)。Dornelas和Rodriguez
(2006)运用Northern杂交和原位杂交技术研究了雪松
(Cedrus deodara)FLORICAULA/LEAFY同源基因
CfLFY的表达量, 发现CfLFY集中在花分生组织和花
芽中表达, 参与了雪松的开花调控。
1.3 抗病虫害类基因
将抗虫基因引入树木细胞并使其在受体细胞内稳定
地遗传和表达, 使林木树种获得对昆虫的抗性, 成为
树木抗虫育种的一种新手段。林业上应用较为普遍的
抗虫基因, 常见的有Bt毒蛋白基因、蛋白酶抑制剂PI
基因和昆虫神经蝎毒素AaIT基因等, 部分抗虫转基
因林木进入田间实验后不可避免地存在生态风险等
问题。林木自身同样具备抗虫基因资源, 近年从喜树
(Camptotheca acuminata)、毛果杨×美洲黑杨杂交杨
(Populus trichocarpa × Populus deltoides)中分离的
TDC、win3等基因显示出抗虫功能。López-Meyer和
Nessler(1997)将喜树的色氨酸脱羧酶基因TDC转化
到欧洲山杨×银白杨杂交杨(Populus tremula ×
Populus alba)中, 过量表达TDC(35S::TDC)的转基
因植株叶片色胺积累, 对天幕毛虫的侵害起到很好的
阻抑作用。win3的编码产物与豆荚种子Kunitz型胰
蛋白酶抑制剂有很高的相似性, 可能是一种胰蛋白

李少锋等: 林木基因克隆研究进展 81
表1 林木功能基因
Table 1 Functional genes of forest trees
种名 基因 功能 参考文献
银白杨(Populus alba L.) CWPO-C 木材形成 Sasaki et al., 2006
银白杨 PaPopCel1 木材形成 Hartati et al., 2008
欧美杨107 (Populus × euramericana ‘74/76’) PAL 木材形成 薛永常等, 2004
欧美杨107 c3h 木材形成 聂会忠和薛永常, 2008
毛白杨(Populus tomentosa Carr.) PtoCesA1 木材形成 李春秀等, 2007
毛白杨 PtoCDKB, PtoCYCB 木材形成 Li et al., 2009
毛白杨 CCoAOMT 木材形成 魏建华等, 2001b
毛白杨 PtEXP1 木材形成 张春玲等, 2006
美洲山杨(Populus tremuloides Michx.) PtSAD 木材形成 Li et al., 2001
美洲山杨 PtrCesA1, PtrCesA2 木材形成 Samuga and Joshi, 2002
美洲山杨 PtrCesA3, PtrCesA4 木材形成 Kalluri and Joshi, 2004
美洲山杨 PtrKOR 木材形成 Bhandari et al., 2006
美洲山杨 Ptomt1 木材形成 Bugos et al., 1991
美洲山杨 CslD 木材形成 Liang and Joshi, 2004
美洲山杨 Pt4CL1, Pt4CL2 木材形成 Hu et al., 1998
美洲山杨 PtrCesA5 木材形成 Kalluri and Joshi, 2003
美洲山杨 CCR 木材形成 Li et al., 2005
美洲山杨 PtrCSLD2 木材形成 Samuga and Joshi, 2004
美洲山杨 PTM5 木材形成 Cseke et al., 2003b
毛果杨(Populus trichocarpa Torr. & Gray) PXP1, PXP2, PXP3, PXP4,
PXP5, PXP6
木材形成 Christensen et al., 1998
毛果杨 PopF5H 木材形成 Sibout et al., 2002
毛果杨 PoptrMP1, PoptrMP2 木材形成 Johnson and Douglas, 2007
毛果杨 PtCOMT 木材形成 Bhuiya and Liu, 2009
毛果杨 PtreC4H, PtriC4H 木材形成 Lu et al., 2006
毛果杨 PtrMYB3, PtrMYB20 木材形成 McCarthy et al., 2010
毛果杨 PtrWND2B, PtrWND6B 木材形成 Zhong et al., 2010
银白杨×大齿杨杂交杨(Populus alba L.× Populus
grandidentata Michx.)
Pa x gINV2, Pa x gINV3 木材形成 Canam et al., 2008
美洲黑杨×毛果杨杂交杨(Populus deltoides Marsh.
× Populus trichocarpa Torr. & Gray)
4CL 木材形成 Allina et al., 1998
日本山杨×大齿杨杂交杨(Populus sieboldii Miquel
apud Hattori & Fujita × Populus grandidentata
Michx.)
palg1, palg2a, palg2b, palg4 木材形成 Osakabe et al., 1995
欧洲山杨×银白杨杂交杨(Populus tremula L.×
Populus alba L.)
ARBORKNOX1 木材形成 Groover et al., 2006
欧洲山杨×银白杨杂交杨 PtaHB1 木材形成 Ko et al., 2005
欧洲山杨×银白杨杂交杨 PtaBXL1 木材形成 Decou et al., 2009
欧洲山杨×银白杨杂交杨 PopFLAs 木材形成 Lafarguette et al., 2004
欧洲山杨×银白杨杂交杨 PtaERF1, PtaRHE1 木材形成 Raemdonck et al., 2005
欧洲山杨×美洲山杨杂交杨(Populus tremula L.×
Populus tremuloides Michx.)
UGDH 木材形成 Johansson et al., 2002
欧洲山杨×美洲山杨杂交杨 PttMYB21a 木材形成 Karpinska et al., 2004
欧洲山杨×美洲山杨杂交杨 PttIAA1–PttIAA8 木材形成 Moyle et al., 2002
欧洲山杨×美洲山杨杂交杨 PttXET16A 木材形成 Kallas et al., 2005
毛果杨×美洲黑杨杂交杨(Populus trichocarpa Torr.
& Gray × Populus deltoides Marsh.)
CAD 木材形成 Doorsselaere et al., 1995
毛果杨×美洲黑杨杂交杨 C4H 木材形成 Ro et al., 2001

82 植物学报 46(1) 2011
表1 (续) Table 1 (continued)
种名 基因 功能 参考文献
美洲黑杨(Populus deltoides Marsh.) PdCO1, PdCO2 花发育 Yuceer et al., 2002
美洲黑杨 FT2 花发育 Hsu et al., 2006
美洲黑杨 PdPI 花发育 Zhang et al., 2008a
毛白杨 PtLFY 花发育 An et al., 2005
毛白杨 PtAP3 花发育 王冬梅等, 2005
欧洲山杨(Populus tremula L.) phyB2 花发育 Ingvarsson et al., 2006
美洲山杨 PTM1, PTM2 花发育 Cseke et al., 2003a
美洲山杨 PTM3/4, PTM6 花发育 Cseke et al., 2005
美洲山杨 MYB134 花发育 Mellway et al., 2009
毛果杨 PTLF 花发育 Rottmann et al., 2000
毛果杨 PtFT1 花发育 Böhlenius et al., 2006
毛果杨 PTAG1, PTAG2 花发育 Brunner et al., 2000
甜杨(Populus suaveolens Fisch.) PsG6PDH 抗冻 林元震, 2006
甜杨 ICE1 抗冻 林元震等, 2007
毛白杨 PtFAD2 抗冻 Zhou et al., 2007a
毛白杨 PtFAD3 抗冻 周洲和李永丽, 2009
胡杨(Populus euphratica Oliv.) PSTZ 抗旱 Wang et al., 2008a
欧美杨(Populus euramericana ‘Dorskamp’) Peudhn1 抗旱 Caruso et al., 2002
河北杨(Populus hopeiensis Hu et Chow) PhCBF4a, PhCBF4b 抗旱 Wang et al., 2008b
毛果杨 PtSRK2C-1, PtSRK2C-2,
PtSRK2C-3
抗旱 毕毓芳等, 2009
小叶杨(Populus simonii Carr.) CMO 抗旱 张守攻等, 2006
新疆杨(Populus alba L. var. pyramidalis) PaTI1 抗虫 梁文星等, 2006
毛白杨 CPI 抗虫 张星耀等, 2007
毛果杨×美洲黑杨杂交杨 KTI3, CHI, PPO1 抗虫 Ramírez et al., 2009
毛果杨×美洲黑杨杂交杨 win3 抗虫 Bradshaw et al., 1989
毛白杨 PtDRG01 抗病 李琰等, 2008
银白杨×欧洲山杨杂交杨
(Populus alba L. × Populus tremula L.)
PoPOD1 抗病 Bae et al., 2006
毛果杨×美洲黑杨杂交杨 win6, win8 抗病 Davis et al., 1991
钻天杨(Populus nigra L. var. italica) PnLPK 机械损伤防护 Nishiguchi et al., 2002
美洲山杨 PtSodCcl 抗氧化 Akkapeddi et al., 1999
美洲山杨 Cu/Zn-sod 抗氧化 Akkapeddi et al., 1994
银白杨 PaMT1, PaMT2, PaMT3 耐重金属污染 Castiglione et al., 2007
毛果杨×美洲黑杨杂交杨 PtdMTP1 耐重金属污染 Blaudez et al., 2003
挪威云杉(Picea abies (L.) Karst.) Cad2, Cad7, Cad8 木材形成 Schubert et al., 2002
扭叶松(Pinus contorta Dougl. ex Loud.) β-glucosidase gene 木材形成 Dharmawardhana et al., 1999
火炬松(Pinus taeda L.) 4CL 木材形成 Zhang and Chiang, 1997
火炬松 PtaAGP6 木材形成 Zhang et al., 2003
火炬松 PtaAGP3 木材形成 Loopstra et al., 2000
火炬松 CesA 木材形成 Nairn and Haselkorn, 2005
火炬松 CCoAOMT 木材形成 Li et al., 1999
火炬松 PtMYB1 木材形成 Patzlaff et al., 2003b
火炬松 PtMYB4 木材形成 Patzlaff et al., 2003a
雪松(Cedrus deodara Loud.) CfLFY 花发育 Dornelas and Rodriguez, 2006
挪威云杉 DAL1 花发育 Carlsbecker et al., 2004
挪威云杉 DAL11, DAL12, DAL13 花发育 Sundstrom et al., 1999
加勒比松(Pinus caribaea Morelet) PcLFY 花发育 Dornelas and Rodriguez, 2005

李少锋等: 林木基因克隆研究进展 83
表1 (续) Table 1 (continued)
种名 基因 功能 参考文献
辐射松(Pinus radiata D. Don) PRFLL 花发育 Mellerowicz et al., 1998
辐射松 NEEDLY 花发育 Mouradov et al., 1998
脂松(Pinus resinosa Ait.) PMADS2 花发育 Liu et al., 2003
白云杉(Picea glauca (Motnch) Voss.) PgDhn1 抗旱 Richard et al., 2000
加拿大短叶松(Pinus banksiana Lamb.) JPD16, JPD18 抗旱 Mayne et al., 1997
火炬松 lp3 抗旱 Padmanabhan et al., 1997
火炬松 pLP2, pLP3, pLP4, pLP5 抗旱 Chang et al., 1996a
西部白松(Pinus monticola Dougl. ex D. Don) PmCh4A 抗病 Liu et al., 2005
西部白松 RGAs 抗病 Liu and Ekramoddoullah, 2003
樟子松(Pinus sylvestris var. mongolica Litv.) PsDef1 抗病 Kovalyova et al., 2007
火炬松 PtTPS-LAS, PtAO 抗病 Ro and Bohlmann, 2006
挪威云杉 Mdh2, Mdh3 抗氧化 Breitenbach-Dorfer and
Geburek, 1995
欧洲赤松(Pinus sylvestris L.) SOD 抗氧化 Karpinska et al., 2001
赤桉(Eucalyptus camaldulensis Dehnh.) Ecliml 木材形成 Kawaoka et al., 2006
赤桉 EcHB1 木材形成 Sonoda et al., 2009
赤桉 Euc4CLE3, Euc4CL1 木材形成 Chen et al., 2006b
巨桉(Eucalyptus grandis Hill ex Maiden) EgraCesA1, EgraCesA2,
EgraCesA3
木材形成 Lu et al., 2008b
巨桉 EgCesA1, EgCesA2,
EgCesA3, EgCesA4,
EgCesA5, EgCesA6
木材形成 Ranik and Myburg, 2006
巨桉 EgMYB2 木材形成 Goicoechea et al., 2005
冈尼桉(Eucalyptus gunnii J.T. Hook) CAD 木材形成 Grima-Pettenati et al., 1993
冈尼桉 EgCCR 木材形成 Lacombe et al., 2000
尾叶桉(Eucalyptus urophylla S.T. Blakely) 4CL 木材形成 孔华等, 2008
蓝桉(Eucalyptus globulus Labill.) EAP1, EAP2 花发育 Kyozuka et al., 1997
蓝桉 ELF1 花发育 Southerton et al., 1998b
巨桉 EgLFY 花发育 Dornelas et al., 2004
巨桉 egm, egm2, egm3 花发育 Southerton et al., 1998a
巨桉 EgrMADS3, EgrMADS4 花发育 Watson and Brill, 2004
蓝桉 EgCBF1 抗冻 Gamboa et al., 2007
冈尼桉 EgSXD1 抗冻 Kayal et al., 2006
冈尼桉 EguCBF1a, EguCBF1b 抗冻 Kayal et al., 2006
巨桉 PGIP 抗病 Chimwamurombe et al., 2001
白桦(Betula platyphylla Suk.) Aux, rol 木材形成 Piispanen et al., 2003
白桦 BXET 木材形成 Toikkanen et al., 2007
白桦 BpSPL1 花发育 Lannenpaa et al., 2004
白桦 BpMADS1, BpMADS6 花发育 Lemmetyinen et al., 2001
白桦 BpMADS3, BpMADS4,
BpMADS5
花发育 Elo et al., 2001
白桦 BpHEN 花发育 杨传平等, 2006
白桦 Bplti36 抗冻 Puhakainen et al., 2004
白桦 BpKASII, BpFAD3,
BpFAD7, BpFAD8
抗冻 Martz et al., 2006
白桦 BpCBF1, BpCBF2,
BpCBF3, BpCBF4
抗冻 Welling and Palva, 2008
毛枝桦(Betula pubescens Ehrh.) BpuDhn, BpuDhn2 抗冻 Welling et al., 2004
盐桦(Betula halophila Ching ex P.C. Li) BhNHX 抗旱 曾幼玲等, 2008

84 植物学报 46(1) 2011
表1 (续) Table 1 (continued)
种名 基因 功能 参考文献
白桦 APX 抗旱 Wang et al., 2009a
白桦 Betv 1-SC1, Betv 1-SC3 抗病 Poupard et al., 1998
白桦 MRP4 耐重金属污染 Keinanen et al., 2007
金丝柳, 爆竹柳
(Salix alba var. tristis Gand, Salix fragilis L.)
C4H 木材形成 Trung et al., 2008
褪色柳(Salix discolor Muhl) SAP1-1, SAP1-2 花发育 Fernando and Zhang, 2006
吉尔吉柳(Salix gilgiana U.V. Seem) pSgPG1, pSgPG2,
pSgPG3, pSgPG4,
pSgPME1, pSgGN1
花发育 Futamura et al., 2000
簸箕柳(Salix suehowensis Cheng) SsMADS 花发育 陈英等, 2007
蒿柳(Salix viminalis L.) Wound-induced proteinase
inhibitor gene
机械损伤防护 Saarikoski et al., 1996
银杏(Ginkgo biloba L.) GbPAL 木材形成 程水源等, 2005
银杏 GbC3H 木材形成 刘学奋, 2006
银杏 GBM5 花发育 Jager et al., 2003
银杏 GinNdly, Ginlfy 花发育 张建业, 2002; 张建业等, 2002
银杏 GbDHN 抗旱 邓仲香, 2006
银杏 Antifungal protein gene 抗病 Sawano et al., 2007
柽柳(Tamarix chinensis Lour.) ThCAP 抗冻 林士杰等, 2006
柽柳 eIF-5A 抗旱 杨传平等, 2005
柽柳 eIF1A 抗旱 高彩球等, 2007
柽柳 LEA 抗旱 Wang et al., 2006
柽柳 TaAQP 抗旱 董玉芝等, 2006
柽柳 Trx 抗旱 王玉成等, 2004
柽柳 dir 抗病 高彩球等, 2006
柽柳 MnSOD 抗氧化 王丙锋等, 2007
柽柳 MT 耐重金属污染 张艳等, 2007c
夜叉桤木(Alnus firma S. et Z (Yashiabushi)) AfHb1 抗氧化 Sasakura et al., 2006
喜树(Camptotheca acuminata Decne.) AOC 抗旱 Pi et al., 2009
喜树 tdc1, tdc2 抗虫 López-Meyer and Nessler,
1997
美国金钟连翘(Forsythia intermedia Zabel) DFR 花发育 Rosati et al., 1997
美国金钟连翘 ANS 花发育 Rosati et al., 1999
欧洲栎(Quercus robur L.) QrDhn1, QrDhn2, QrDhn3 抗旱 Šunderlíková et al., 2009a
欧洲栎 QrchitIII-1, QrchitIII-2 抗病 Frettinger et al., 2006
欧洲栓皮栎(Quercus suber L.) QsCAD1 抗病 Coelho et al., 2006
欧洲水青冈(Fagus sylvatica L.) FsDhn1, FsClo1 抗旱 Jiménez et al., 2008
欧洲榛(Corylus avellana L.) CaMADS1 花发育 Rigola et al., 1998
刺槐(Robinia pseudoacacia L.) RpARP 木材形成 Park and Han, 2003
刺槐 RPNHX1 抗旱 于学宁等, 2007
团花树(Anthocephalus chinensis
(Lam.) A. Rich. et Walp.)
AcEXP1 木材形成 欧阳昆唏等, 2010
落羽杉(Taxodium distichum (Linn.) Rich.) rbcL 抗旱 Soltis et al., 1992


酶抑制剂基因, 能够抑制害虫胰蛋白酶原的激活或
胰脏中其它蛋白酶原的活化 , 干扰害虫的消化吸
收 , 起到防治害虫的目的(Bradshaw et al., 1989)。
林木病害的病原菌种类多, 变异速度快, 分离、
纯化工作不易进行。以前树木病害的研究主要集中在
抗病QTL与抗病质量性状定位, 以及抗病基因相似序
列的克隆与鉴定方面。转基因技术研究主要利用外源
的病毒外壳蛋白基因CP、几丁质酶基因Chi和抗菌肽
李少锋等: 林木基因克隆研究进展 85
表2 林木其它功能基因
Table 2 Other functional genes of forest trees
种名 基因 参考文献
香脂杨(Populus balsamifera L.) RRs Ramirez-Carvajal et al., 2008
美洲黑杨(Populus deltoides Marsh.) POMT-7 Kim et al., 2008a
美洲黑杨 psbD, psbC Reddy et al., 1998
美洲黑杨 psbE-F-L-J Naithani et al., 1997
美洲黑杨 NADP-ME Doorsselaere et al., 1991
美洲黑杨 5.8S rDNA gene DOvidio, 1992
美洲黑杨 PdLOX1, PdLOX2 Cheng et al., 2006
毛白杨(Populus tomentosa Carr.) Wuschel Yang et al., 2009
毛白杨 PtFATB Zhou et al., 2007b
毛白杨 PtCDD Cao et al., 2008
毛果杨(Populus trichocarpa Torr. & Gray) PtIAMT1 Zhao et al., 2007
毛果杨 PtVIN(1–3), PtCIN(1–5), PtNIN(1–16) Bocock et al., 2008
毛果杨 PtCOBRA Ye et al., 2009
毛果杨 CAZymes Geisler-Lee et al., 2006
毛果杨 PtABI3 Rohde et al., 1998
毛果杨 PtSABP2-1, PtSABP2-2 Zhao et al., 2009
毛果杨 PtCBL Zhang et al., 2008b
银白杨×欧洲山杨杂交杨
(Populus alba L. × Populus tremula L.)
Isoprene synthase gene Miller et al., 2001
日本山杨×大齿杨杂交杨(Populus sieboldii Miquel apud
Hattori & Fujita × Populus grandidentata Michx.)
cyp73a, cyp73b, cyp73c Kawai et al., 1996
欧洲山杨×银白杨杂交杨(Populus tremula L. × Populus
alba L.)
PtaZFP2 Martin et al., 2009
欧洲山杨×银白杨杂交杨 ET304 Filichkin et al., 2006
欧洲山杨×银白杨杂交杨 PtaGA2ox1 Busov et al., 2003
欧洲山杨×美洲山杨杂交杨(Populus tremula L. ×
Populus tremuloides Michx.)
GA3ox Israelsson et al., 2004
毛果杨×美洲黑杨杂交杨(Populus trichocarpa Torr. &
Gray × Populus deltoides Marsh.)
PtdPPO2, PtdPPO3 Wang and Constabel, 2004
毛果杨×美洲黑杨杂交杨 PtdLOX1, PtdTPS1 Arimura et al., 2004
毛果杨×美洲黑杨杂交杨 BSP, WIN4, pni288 Lawrence et al., 2001
美洲落叶松(Larix laricina (Du Roi) Koch.) rbcS Hutchison et al., 1990
挪威云杉(Picea abies (L.) Karst.) Chia4-Pa1 Wiweger et al., 2003
挪威云杉 HBK1 Sundas-Larsson et al., 1998
挪威云杉 Pavp1, cdc2Pa Footitt et al., 2003
挪威云杉 sHSP Schubert et al., 2002
挪威云杉 PaVP1 Fischerova et al., 2008
挪威云杉 Pa18 Sabala et al., 2000
挪威云杉 His2A Sundas and Engström, 1995
挪威云杉 PaMST1 Nehls et al., 2000
挪威云杉 ABI3 Lazarova et al., 2002
加勒比松(Pinus caribaea Morelet) PcGER1 Neutelings et al., 1998
加州山松(Pinus monticola Dougl. ex D. Don) TIR-NBS-LRR resistance gene analogs Liu and Ekramoddoullah, 2003
加州山松 PmWRKY Liu and Ekramoddoullah, 2009
沙地海岸松(Pinus pinaster Ait.) PpAAI-LTSS1, PpRab1, PpCR1 Goncalves et al., 2005
辐射松(Pinus radiata D. Don) 5S rDNA genes Moran et al., 1992
美国五针松(Pinus strobus L.) Glb-2 Baker et al., 1996

86 植物学报 46(1) 2011
表2 (续) Table 2 (continued)
种名 基因 参考文献
欧洲赤松(Pinus sylvestris L.) PsRLK Avila et al., 2006
欧洲赤松 Glutamine synthetase gene Canton et al., 1993
欧洲赤松 Lhcb5 Jansson and Gustafsson, 1994b
欧洲赤松 PsAS1 Cañas et al., 2006
樟子松(Pinus sylvestris var. mongolica Litv.) GS1a Avila et al., 2002
樟子松 Psnir Neininger et al., 1994
樟子松 Lhca4.1 Jansson and Gustafsson, 1994a
樟子松 PsySCR Laajanen et al., 2007
火炬松(Pinus taeda L.) pLP6 Chang et al., 1996b
火炬松 PTIAA1, PTIAA2, PTIAA3, PTIAA4, PTIAA5 Goldfarb et al., 2003
火炬松 5NG4 Busov et al., 2004
火炬松 PtIDS1, PtIDS2 Kim et al., 2008c
黑松(Pinus thunbergii Parl) Ribulose bisphosphate carboxylase small
subunit gene
Yamamoto et al., 1988a
黑松 Light harvesting chlorophyll a/b binding
protein gene
Yamamoto et al., 1988b
黑松 LCHPII Yamamoto et al., 1993
花旗松(Pseudotsuga menziesii (Mirbel) Franco) rbcL Hipkins et al., 1990
花旗松 PmTLP Zamani et al., 2004
花旗松 ACS1, ACS2, ACS3, ACS4 Ralph et al., 2007
赤桉(Eucalyptus camaldulensis Dehnh.) EcPT1, EcPT2, EcPT3, EcPT4, EcPT5 Koyama et al., 2006
蓝桉(Eucalyptus globulus Labill.) ETL Decroocq et al., 1999
蓝桉 EgHypar Nehls et al., 1998
白桦(Betula platyphylla Suk.) SAGs, Pr1 Sillanpää et al., 2005
白桦 nia Hachtel and Strater, 2000
白桦 BpMADS2 Jarvinen et al., 2003
白桦 Hexose transporter gene, sucrose
transporter gene
Wright et al., 2001
白桦 RbcS Valjakka et al., 1999
白桦 BP8 Puupponen-Pimia et al., 1993
白桦 Mpt1 Kiiskinen et al., 1997
纸皮桦(Betula papyrifera L.) 5S rRNA genes Johnson et al., 1992
吉尔吉柳(Salix gilgiana U.V.Seem) pSgPEL1 Futamura et al., 2002
吉尔吉柳 pSGJ1, pSGJ2, pSGJ3 Futamura et al., 1999
银杏(Ginkgo biloba L.) GbIDS1, GbIDS2, GbIDS2-1 Kim et al., 2008c
银杏 GbMECS, GbDXS1, GbDXS2 Kim et al., 2006c
银杏 PBR Amri et al., 2002
银杏 GbLeg1, GbLeg2 Hager et al., 1995
银杏 GBE Shinozuka et al., 2002
银杏 chlB Richard et al., 1994
银杏 GbCMK1, GbCMK2 Kim et al., 2008b
银杏 GbMECT Kim et al., 2006a
银杏 GbHDR Lu et al., 2008a
银杏 GbIspF 彭梅芳等, 2008
银杏 Lacasse gene 徐小勇和杨甜甜, 2009
银杏 GGDPS Wang et al., 2009b
银杏 GbAsr Shen et al., 2005a
银杏 GbANS Xu et al., 2008b

李少锋等: 林木基因克隆研究进展 87
表2 (续) Table 2 (continued)
种名 基因 参考文献
银杏 GbatpA, GbatpB Xu et al., 2008a
银杏 GbGST Liu et al., 2007
银杏 GbHMGR, GbANR Shen et al., 2006
银杏 Gbd Shen et al., 2005b
银杏 GbCHS Pang et al., 2005
银杏 GbMECPS Gao et al., 2006
银杏 GbDXS Gong et al., 2006
银杏 GbDXR Kim et al., 2006b
银杏 GbGGPPS Liao et al., 2004
银杏 Gbchs2 Xu et al., 2007
柽柳(Tamarix chinensis Lour.) E2s 徐晨曦等, 2007
柽柳 GST 杨平等, 2007
欧洲桤木(Alnus glutinosa (L.) Gaertn.) Nodule-specific gene Pawlowski et al., 1997
灰赤杨(Alnus incana L.) Chloroplast 23S rRNA gene Gutell et al., 1992
灰赤杨 5S rRNA gene Johnson et al., 1992
喜树(Camptotheca acuminata Decne.) hmg1, hmg2, hmg3 Maldonado-Mendoza et al., 1997
喜树 DXR Yao et al., 2008
喜树 TSB Lu and McKnight, 1999
喜树 CAP19 Maldonado-Mendoza
and Nessler, 1996
喜树 CaASA1, CaASA2 Lu et al., 2005
樟树(Cinnamomum camphora (L.) Presl) Cin c1 宋娟娟等, 2006
樟树 Genes encoding cinnamomin (a type II RIP) Yang et al., 2002
黄扁柏(Chamaecyparis nootkatensis (D. Don) Spach) CnABI3 Lazarova et al., 2002
桂花(Osmanthus fragrans Lour.) OfCCD4 Huang et al., 2009
金桂(Osmanthus fragrans var. thunbergii Makino) OfLis 唐丽等, 2009
桑树(Morus alba L.) MaACO1 Pan and Lou, 2008
桑树 MPP Sharma et al., 2001
桑树 Mahmg1 Jain et al., 2000
栓皮栎(Quercus variabilis Blume.) B-type cyclin gene Neves et al., 2006
大果栎(Quercus macrocarpa Michx.) Four putative aquaporin genes Voicu et al., 2009
欧洲栎(Quercus robur L.) QrEm Šunderlíková et al., 2009b
无梗花栎(Quercus petraea (Matt.) Liebl.) QpHb1 Parent et al., 2008
欧洲水青冈(Fagus sylvatica L.) DAHPS1, DAHPS3, DHQD, SD, DAHPS2,
EPSPS, CM, DHQS, SK, CS, GA
Betz et al., 2009
欧洲水青冈 A small GTP-binding protein gene Nicolás et al., 1998
欧洲水青冈 FsPP2C2 Reyes et al., 2006
欧洲水青冈 FsPP2C1 González-García et al., 2003
北美鹅掌楸(Liriodendron tulipifera L.) Ltlacc2.1, Ltlacc 2.2, Ltlacc2.3, Ltlacc2.4 LaFayette et al., 1999


基因LCI等, 导入目标树种中, 以提高林木的抗病性。
近年来从樟子松(Pinus sylvestris var. mongolica)、
加州山松 (Pinus monticola)和火炬松中克隆了
PsDef1、PmCh4A、PtTPS-LAS和PtAO等抗病毒基
因 , 大大促进了林木抗病基因工程的研究进程。
Kovalyova等(2007)从樟子松中分离获得了长度为
252 bp的防御素基因PsDef1, 其具有广谱、高效的抗
微生物活性。几丁质酶是植物抗真菌酶, 外源茉莉酮
酸、蛋白磷酸酶1A、2A抑制剂处理和机械损伤均导
致加州山松中PmCh4A蛋白的积累增加, 显示了几丁
质酶基因PmCh4A在防御疱锈病和非生物胁迫方面
的重要作用(Liu et al., 2005)。Ro和Bohlmann (2006)
88 植物学报 46(1) 2011
从火炬松中分离了PtTPS-LAS基因, 其编码二萜合
酶。二萜合酶是二萜醌类化合物生物合成的一个关键
酶。萜类化合物具有抑制真菌活性的作用, 可以避免
林木受到真菌感染。
1.4 抗逆基因
在干旱胁迫下林木自身的调控基因和功能基因均被
诱导表达。调控基因编码DREB、MYB/MYC和bZIP
等转录因子, 它们与响应水分胁迫的顺式作用元件相
结合, 从而激活下游基因表达。渗透调节物质——蔗
糖、脯氨酸、水通道蛋白和LEA蛋白等属于功能性基
因的表达产物, 可直接参与减轻干旱胁迫所造成的危
害。干旱胁迫应答基因编码蛋白——LEA蛋白是一类
重要的脱水素, 具有高度亲水性, 能够保护细胞免受
水分胁迫的伤害。欧洲栎(Quercus robur)和欧美杨
(Populus euramericana ‘Dorskamp’)的脱水素基因
QrDhn1、QrDhn2、QrDhn3、peudhn1均能够响应
外部缺水条件, 其表达量明显提高, 具有增强植物脱
水耐性的功能(Caruso et al., 2002; Šunderlíková et
al., 2009a)。Mayne等(1997)报道了加拿大短叶松
(Pinus banksiana)干旱诱导基因JPD16和JPD18, 在
外源10 λmol·m–3ABA作用1小时后, 其在植株根和针
叶部的表达量明显上升。Chang等(1996a)从遭受水
分胁迫的火炬松根部cDNA文库中分离到pLP5, 该基
因可能编码细胞壁增厚蛋白, 防止植株脱水萎蔫。欧
洲水青冈(Fagus sylvatica)ABA响应基因FsDhn1编
码晚期胚胎富集蛋白LEA, 在欧洲水青冈种子干燥的
条件下, LEA蛋白受诱导特异表达, 表明FsDhn1 是
一个干旱诱导基因 (Jiménez et al., 2008)。喜树
CaAOC基因受盐环境诱导表达, 原核表达实验表明,
转化子能在400 mmol·L–1 NaCl的高盐培养基中正常
生长, 且盐胁迫下转基因烟草的叶绿素含量明显高于
对照(Pi et al., 2009)。
植物适应低温反应的产生有依赖ABA和非依赖
ABA两种途径。前者包括ABA应答元件和带有bzip基
序的ABA应答元件结合蛋白; 后者包括CRT/DRE顺
式作用元件和CBF结合蛋白(CRT/DRE-binding fac-
tor)等重要构件。Kayal等(2006)从冈尼桉(Eucalyptus
gunnii)分离的冷诱导基因EguCBF1a和EguCBF1b,
编码CRT/DRE结合因子, 能够响应短日照和低温胁
迫。Puhakainen等(2004)从白桦中克隆获得低温诱导
基因Bplti36, 并发现在短日照和低温处理下, Bplti36
转录水平显著上升。白桦脂肪酸生物合成基因
BpFAD3、BpFAD7和BpFAD8, 参与亚油酸(18:2)转
变为α-亚麻酸(18:3)的过程; 低温能诱导BpFAD3和
BpFAD8的表达, 但抑制BpFAD7的表达, 使α-亚麻
酸(18:3)含量增高, 脂肪酸不饱和性增加, 从而提高
白桦的抗寒能力(Martz et al., 2006)。
逆境胁迫下植物的抗氧化系统主要由超氧化物
歧化酶(SOD)、过氧化物酶(POD)和谷胱甘肽(GSH)
等抗氧化物组成, 它们协同作用抵抗活性氧对植物机
体的伤害。其中关键的抗氧化酶基因——铜/锌超氧化
物歧化酶基因PtSodCcl, 由Akkapeddi等(1999)从臭
氧胁迫的美洲山杨叶片基因组中克隆, Northern杂交
结果显示, PtSodCcl对臭氧诱导敏感, 其表达量迅速
上升, 推测其能够清除臭氧的胁迫毒害。夜叉桤木
(Alnus firma)血红素Hb基因AfHb1受NO诱导, 促使
NO清除剂的产生, 从而降低NO胁迫对机体的损害
(Sasakura et al., 2006)。此外, Sillanpää等(2005)的
研究认为, 半胱氨酸蛋白酶基因Cyp1和Cyp2参与白
桦叶片衰老的调控, 是一种重要的抗氧化物质。
当今环境污染日趋严重, 树木自身具备一定的
修复功能 , 对维持生态平衡和抵抗环境恶化具有重
要作用。近年来, 基因工程技术逐渐成为对被污染环
境进行生物治理的有力工具。从杨树中分离的
PtdMTP1、白桦的MRP4等基因及其应用, 对于阐明
树木抗环境污染的机制有重要意义。PtdMTP1基因从
毛果杨×美洲黑杨杂交杨中克隆得到, 具有解除Cd和
Zn胁迫的能力, 并对抑制Co、Mn和Ni等重金属的富
集有一定效果(Blaudez et al., 2003)。Keinanen等
(2007)运用抑制消减杂交(SSH)技术从Cu胁迫的白
桦中获得耐重金属基因MRP4, 其在根与芽部的表达
量显著上升, 参与消除重金属Cu对生物的毒性。
2 国内林木基因克隆研究概况
我国自20世纪80年代末期开始林木转基因的研究,
主要采用叶盘法和基因枪等转化方法, 将农作物或细
菌的基因转化至林木基因组中。利用林木基因进行遗
传改良虽然起步较晚, 但发展迅速, 已获得具有潜在
应用价值的新基因80多个, 其中参与木材形成的有
15个, 花发育7个, 抗冻4个, 抗旱15个, 抗病2个,
李少锋等: 林木基因克隆研究进展 89
抗虫2个, 抗氧化1个, 耐重金属污染3个, 其它功能
基因35个。上述各基因及其功能详见表1和表2。
2.1 与材质有关的基因
国内科研人员从欧美杨107(Populus x euramericana
‘74/76’)、毛白杨 (Populus tomentosa)、尾叶桉
(Eucalyptus urophylla)中分离克隆了PAL、CCo-
AOMT、4CL、C3H及COMT等参与木质素生物合成
的关键基因, 为利用基因工程调控林木木质素的研究
奠定了基础(魏建华等 , 2001a, 2001b; 薛永常等 ,
2004; 孔华等, 2008; 聂会忠和薛永常, 2008)。另外,
Li等(2009)的研究认为, 细胞周期蛋白依赖性激酶基
因CDKB和CYCB参与毛白杨形成层细胞分裂。扩展
蛋白expansin是植物细胞壁松弛因子。张春玲等
(2006)利用RT-PCR从毛白杨树干形成层部位克隆了
编码该蛋白的PtEXP1基因, PtEXP1可能参与形成层
细胞向木质部细胞的分化。欧阳昆唏等(2010)采用
RACE技术, 以团花树(Anthocephalus chinensis)枝
条形成层的cDNA为模板, 克隆得到扩展蛋白全长基
因AcEXP1。李春秀等(2007)从毛白杨中克隆获得长
度为 3 215 bp的纤维素合成酶基因PtoCesA1;
35S::PtoCesA1转基因烟草株系生长受到抑制, 其基
部木质部厚度比对照植株和野生型植株小, 纤维细胞
壁厚度也有所减小, 表现出反义抑制的表型, 可能发
生了转录后基因沉默 (post-transcriptional gene
silencing, PTGS)现象。贺郭等(2010)根据毛果杨
NAC068基因设计引物, 从毛白杨基因组中克隆得到
该基因5’侧翼区901 bp长的片段pProNAC068; 该启
动子介导的GUS活性检测表明, 其在形成层区域启
动表达的强度比35S启动子的高。
2.2 与生殖有关的基因
培育不育的转基因林木新品种可以有效减少转基因
花粉引起的基因逃逸。例如王冬梅等(2005)构建了毛
白杨PtAP3基因的正反义表达载体, 并获得转基因烟
草植株, 下一步将开展毛白杨的遗传转化工作, 以期
实现干扰毛白杨等树种的开花, 使花器官败育, 从而
控制毛白杨飞絮污染。PtAP3具有高度保守的
MADS-box区, 在毛白杨花发育调控过程中作为转录
因子发挥作用。苏晓华等(2009)从美洲黑杨(Populus
deltoides)雄性花芽cDNA文库中分离得到MADS-box
基因PdPI, 其在花蕾的表达量明显高于其它各组织
部位; 转基因研究结果表明, 转正义PdPI基因的烟草
植株提前开花, 而转反义基因的植株与对照相比无明
显差异。PdPI在花蕾、雄花序的花被和花药中表达量
高, 在花梗和成熟花粉中的表达量低, 与花器官的发
育和开花调控密切相关(Zhang et al., 2008a)。安新民
等(2010)从毛白杨花芽中克隆了PtLFY cDNA序列,
其在雌雄花芽发育过程中的差异表达表明PtLFY参与
花芽的形态分化。GinNdly是花分生组织基因LEAFY
的同源基因。张建业等(2005)利用银杏GinNdly基因
构建了正义与反义植物表达载体, 转基因的研究还在
进行当中。
2.3 抗病虫害类基因
病毒、真菌和细菌有着多变的致病机制, 由它们引起
的林木病害相当严重, 每年因森林病虫害造成的经济
损失高达数十亿元。相比农作物, 树木抗病基因的研
究较缺乏, 目前国内克隆的抗病基因仅有PtDRG01
和 dir。李琰等 (2008)构建了 NBS型抗病基因
PtDRG01的原核表达载体, 导入大肠杆菌, 经IPTG
诱导表达获得目标长度的特异表达蛋白, 为下一步的
蛋白功能研究奠定了基础。李海霞等(2009)将毛白杨
的PtDRG01基因导入烟草, 转基因烟草株系TG-11
叶片中的烟草花叶病毒(TMV)数量显著少于非转基因
植株, 表明PtDRG01基因具有抗病毒功能。高彩球等
(2006)通过构建柽柳cDNA文库 , 挑取单克隆测序 ,
获得dir基因的cDNA全长序列; dir基因编码产物参与
木质素的合成, 与柽柳的病菌防御有关。
天牛可对多种林木造成危害, 杨树、柳树、槐树
等均受其直接威胁, 林区受害面积大, 造成的经济损
失惨重。对此, 相关专家开展了一系列研究。张星耀
等(2007)将来源于河北毛白杨形成层的巯基蛋白酶
抑制剂基因CPI, 转入到天牛易感树种中, 直接影响
天牛的取食和消化, 甚至杀死害虫。win3基因编码产
物类似白薯或豆类胰蛋白酶抑制剂, 李强等(1999)从
欧洲黑杨(Populus nigra)和美洲黑杨中扩增出win3
基因启动子WINP和WIDP, 两者分别控制的GUS基
因表达具有因伤诱导的效应。梁文星等(2006)运用
RT-PCR方法从新疆杨(Populus alba var. pyrami-
dalis)叶片中克隆到1个损伤诱导型Kunitz胰蛋白酶抑
制剂基因PaTI1。原核表达实验表明, 纯化后的融合
90 植物学报 46(1) 2011
蛋白对胰蛋白酶的活性有抑制作用, 每8.5 μg融合蛋
白可完全抑制1 μg牛胰蛋白酶的活性, 可有效减弱或
阻断蛋白酶对外源蛋白质的水解作用。
2.4 抗逆基因
冷适应蛋白是低温诱导表达的一类特殊蛋白质。林士
杰等(2006)从柽柳cDNA文库中分离得到冷适应蛋白
基因ThCAP片段 , 并应用RACE技术获得其全长
cDNA序列。Guo等(2009)在进一步的转基因研究中,
获得转基因(35S::ThCAP)山新杨(Populus davidiana
× Populus bolleana), 转基因植株对低温的抵抗能力
明显增强, 证实ThCAP是一个重要的耐寒基因。甜杨
(Populus suaveolens)冷诱导基因ICE1的编码产物
ICE, 是在低温时诱导抗冻因子CBF家族表达的转录
激活因子, 可特异性结合CBF3启动子的MYC作用元
件, 启动CBF3基因的表达; 然后CBF3转录激活因子
再结合到其下游目的基因启动子的DRE/CRT序列上,
诱导抗冻基因COR的表达, 从而提高植株的抗冻性
(林元震等, 2007)。Lin等(2005)对甜杨PsG6PDH基因
的原核表达实验表明, 受1 mmol·L–1 IPTG诱导4小
时, 融合蛋白具有可溶性, 且没有向细胞外分泌, 表
明该基因在维持细胞渗透势、参与抗冻胁迫中起重要
作用。
我国西部和北部的广阔区域长期面临严重的缺
水威胁。如何从林木自身分离优良抗旱基因, 用于抗
旱耐盐节水新品种的培育, 解决西部和北部干旱半干
旱地区大面积造林问题, 已成为当前需要完成的重大
课题。国内专家对此展开了一系列研究。Chen等
(2009)从旱生树种胡杨(Populus euphratica)中分离
到脱水响应结合元件PeDREB2, 转基因烟草对盐生
环境的适应性得到改善, 同时其生长没有受到影响。
Wang等(2009a)从白桦cDNA文库中克隆了编码区长
度为750 bp的抗坏血酸过氧化物酶APX基因, 荧光
实时定量PCR分析显示, 在盐胁迫状态下, APX被高
丰度诱导表达, 可能参与白桦的抗盐机制。张守攻等
(2006)将小叶杨(Populus simonii)胆碱单氧化物酶基
因CMO整合至杂种落叶松 (Larix leptolepis × L.
olgensis)基因组中, 基因表达和功能检测证实, 杂种
落叶松的抗旱能力得到了明显的提高。这些研究对阐
明林木抗旱或耐盐机制, 实现转基因林木抗旱新品系
的选育有重要价值。
在遭遇环境胁迫时, 树木依赖抗氧化防御系统,
防止膜脂过氧化作用和清除超氧阴离子的攻击, 从而
保护植株自身免受活性氧的伤害。王丙锋等(2007)利
用RACE技术从柽柳中获得MnSOD的cDNA全长序
列, 转化至酵母基因组后, 重组酵母的抗干旱、抗高
温能力均明显高于对照非重组酵母。重金属是造成土
壤和水系统污染的重要因素, 利用林木对重金属进行
吸收和转运, 能有效降低土壤重金属的含量, 达到环
境修复的目的。张艳等(2007a)将柽柳的金属硫蛋白
基因MT2连入载体pROKII, 抗性实验证明, 转基因
烟草对Cd2+的抗性显著提高。另外, 将柽柳金属硫蛋
白基因MT1导入烟草 , 发现在含有200 μmol·L–1和
400 μmol·L–1Cd2+的MS培养基上, 过量表达MT1转
基因烟草的株高和鲜重均明显优于非转基因对照植
株, 且叶绿素含量和SOD活性明显增加, 表明MT1是
一个介导Cd2+胁迫抗性的优良基因(张艳等, 2007b)。
3 林木基因克隆及应用研究展望
目前, 由于对不同树种遗传背景的了解程度有差异,
因此需要选择性地采取不同克隆方法和策略。如果已
知目的基因的全部或部分序列, 可通过电子克隆、
RACE或染色体步移技术分离基因全长。也可以针对
已知目的蛋白的某些氨基酸信息, 设计合适的寡核苷
酸探针(库), 通过与含该基因信息的基因组文库或
cDNA文库杂交 , 分离编码此蛋白的DNA序列或
cDNA片段; 或设计目的蛋白的特异抗体与靶蛋白专
一结合, 从基因表达文库中分离目的蛋白基因。对于
不同组织、器官中不同胁迫状态下差异性表达的基因,
可以选用差异显示反转录PCR(DDRT-PCR)、RNA指
纹技术(RAP-PCR)和外显子捕获等克隆方法。当目标
基因的时空表达没有差异, 或没有任何表达功能信息
时, 常采用构建文库直接测序、图位克隆法或转座子
标签法等实现目标基因的克隆。
总体上看, 林木材质形成、开花调控和抗旱相关
的基因克隆研究有一定基础, 但抗冻、抗病虫害、抗
环境污染等方面的研究还很薄弱。我国林木资源虽然
丰富, 但缺少对速生、优质、抗逆等优良特性的乡土
树种的发现和挖掘, 这直接造成了与林木优质高抗相
关的主效基因的匮乏。已经克隆获得的林木抗逆功能
基因, 没有迅速有效地进行遗传转化并加以利用。另
李少锋等: 林木基因克隆研究进展 91
外, 基因克隆的方法单一, 绝大部分目标基因的分离
仅限于RT-PCR和RACE技术, 高效实用的新方法如
TAIL-PCR、MOPAC、cDNA捕捉法等并未得到广泛
应用。此外, 在克隆策略的选择上针对性不强, 盲目
地进行目标基因的大通量分离, 反而容易事倍功半。
目前, 林木转基因主要采用农杆菌侵染法和基因
枪法。前者易发生单拷贝或低拷贝的转化事件, 而且
不适于银杏、杉木、松柏类等树种的遗传转化; 后者
的转化效率同样不高, 且轰击过程中可能造成外源基
因的断裂, 导致插入基因失活。目前报道的转基因手
段还难以做到将转入的基因定点、定量地整合到宿主
基因组中。在植物表达载体的构建中, 大多数使用
CaMV 35S组成型启动子, 对诱导型启动子或组织特
异型启动子欠缺考虑。外源基因导入植物细胞后, 插
入到不同的染色体, 整合到不同的DNA区段(整合位
点), 而且整合的拷贝数和次数也有不同。发生整合的
基因是否具有完整性及是否影响到整合位点的DNA
结构, 在林木中是否同样遵循孟德尔遗传规律, 以及
外源基因在林木子代中传递的稳定性等, 这些都可能
直接影响外源基因功能的鉴定和验证。利用基因工程
技术对林木进行改良, 虽然已取得飞速的发展, 并展
现着诱人的发展前景, 但不能因此取代常规育种, 新
种质的创造、新品种的选育有赖于两者的有机结合。
针对林木本身的特点和研究现状, 我们认为今后
在林木基因克隆及应用方面需加强以下几个方面的
研究。
(1) 选择更新更强的组织特异型启动子或诱导型
启动子, 控制转入基因的表达量、表达时间和表达部
位。在木材发育研究中, 利用林木中分离的PAL、
CAD、CCoAOMT、4CL、pProNAC068等维管组织
特异启动子代替35S启动子, 驱使靶基因特异性地在
维管组织中表达, 可以避免持续、高效表达造成的能
量损耗和转基因安全性问题(Feuillet et al., 1995;
Gray-Mitsumune et al., 1999; Chen et al., 2000; Lu
et al., 2004; Bedon et al., 2009; 贺郭等, 2010)。另
外利用抗逆(如抗病)相关顺式作用元件人工构建理想
启动子, 在农作物抗病研究中已开始起步(Gurr and
Rushton, 2005), 在林业中未见相关报道。
(2) 通过正向遗传学方法, 构建林木例如模式树
种杨树的突变体库(张勇等, 2006), 分离主效基因。利
用基因捕获、T-DNA标签(农杆菌介导的T-DNA插入)、
转座子插入、RNAi、基因敲除等手段构建杨树基因
突变体库, 造成单个基因或基因家族的功能抑制、缺
失或激活。根据单株表型鉴别、理化和材性分析等手
段, 鉴定和克隆关键的主效基因。在后续的研究中,
可以开展基因的功能互补实验, 以进一步验证目标基
因的功能。目前, 拟南芥突变体诱导和大规模的突变
体库筛选已实现完全自动化。国外正有效地开展树木
突变体库的研究, 国内对此的关注度不太高, 因此需
要在该领域加强研究。
(3) 选择新的克隆方法。基于表达序列标签
(expressed sequence tag, EST)方法的基础, 近几年
基因表达系列分析技术 (serial analysis of gene
expression, SAGE)得到了发展和完善, 能够分离差
异表达的基因, 并对上千个转录产物进行定性和定量
分析, 并在火炬松木质部轴向梯度发育的差异基因表
达研究中得到应用(Lorenz and Dean, 2002)。靶向克
隆法是一种新的基因克隆方法, 其使用新开发的靶向
克隆酶LP Recco, 能够使末端序列相同的双链DNA
(14–18 bp)同源重组, 从而达到连接克隆基因和载体
的目的(俞远东等, 2009)。基因表达指纹图谱(gene
expression fingerprinting, GEF)利用含有标记末端的
片段经凝胶电泳后构成的mRNA指纹图谱, 分析不同
细胞间的指纹图谱从而得到差异表达的序列
(Ivanova and Belyavsky, 1995)。另外, cDNA 3′端限
制酶切片段显示(restriction fragment display, RFD)
技术、分子指数的RNA指纹技术和标签接头竞争PCR
技术(ATAC-PCR)等在人类、酵母、作物等的基因克
隆和表达研究中有一定的应用(Prashar and Weiss-
man, 1996; Kato, 1996, 1997)。这些较新的克隆方法
可以试探性地用于林木基因的分离和表达研究中。
(4) 导入转录因子基因, 提高抗逆性。一个抗逆
转录因子基因可以调控下游一批与抗逆相关的功能
基因的表达。在这方面对于农作物的研究已进行得比
较深入, DREB、CBF、ERF等优良的转录因子基因
已实现有效转化(Ito et al., 2006; Chen et al., 2007;
Tang et al., 2007)。林木中虽然克隆了EguCBF1a、
EguCBF1b、ICE1、PaDREB2、AP2/ERF、PhCBF4a
和PhCBF4b等抗逆转录因子基因(家族)(Nanjo et al.,
2004; Qin et al., 2005; Kayal et al., 2006; 林元震等,
2007; Wang et al., 2008b), 但还未见转基因成功的
报道, 因此基因转化的研究力度需要进一步加大。
92 植物学报 46(1) 2011
(5) 建立多基因转化体系。抗虫、抗病等性状大
多是由多基因控制的, 单个基因的导入在抗虫、抗病
性状改良中起的作用微小, 且害虫易对单个抗性基因
产生耐受性。解决问题的有效途径有2条。(1) 将多个
目标基因直接融合或用调控元件隔开构建在1个表达
载体中。例如Chen等(2006a)利用gateway技术构建
了含有7个外源基因片段的多基因表达载体, 并成功
实现遗传转化, 但在林业中还未见此类报道。(2) 将
多个单价基因分别构建在多个表达载体上, 利用基因
枪等方法实现共同转化。如王建革等(2006)将含有5
个外源基因的 4个表达载体成功导入库安托杨
(Populus × euramericana ‘Guariento’), 中试实验表
明, 多数转基因植株生长状况良好。后一种共转化途
径的共整合频率和共表达频率可能比前者要低。
(6) 完善核酶基因操作和基因转化技术。核酶指
具有催化功能的RNA分子, 能够完成自身的剪接反
应。据此可设计、合成特异性切割病毒RNA的核酶基
因, 导入植物中以验证抗病毒侵染的性能。该技术已
在番茄(Solanum lycopersicum)和马铃薯(Solanum
tuberosum)的抗病研究中得到应用(朱秋菊 , 2005;
张剑峰等, 2008), 但在林木研究中还未见相关报道。
质体基因转化在近年来应用广泛, 成为新的研究热
点。叶绿体转化体系利用叶绿体的同源片段将外源基
因定点整合到植物的叶绿体基因组中, 通过其高拷贝
性实现外源基因的高效表达, 在烟草、大豆(Glycine
max)、胡萝卜 (Daucus carota)和棉花(Gossypium
spp.)等植物中已获得成功转化(Kumar et al., 2004a,
2004b; Dufourmantel et al., 2005; Chakrabarti et
al., 2006), 并提高了烟草、胡萝卜的抗虫、耐盐性。
这种新的转化技术可以尝试用于抗逆、抗病虫林木的
培育。转座子是基因组中一段特异的具有转位特性的
DNA序列, 通过切割、重新整合等方式, 可以从基因
组的一个位置“跳跃”到另一个位置。转座子可以介
导外源基因进入植物细胞中, 并能够在后代中消除选
择标记, 从而免除人们对含有选择标记基因的转基因
产品可能导致环境污染的担忧, 这对转基因林木的环
境释放有一定的借鉴意义。
(7) 重视基因表达策略, 克服转基因沉默。利用
组织和发育特异性的增强子与转入基因构建嵌合体,
能有效去除甲基化作用, 提高基因转录频率和外源基
因的表达水平。将外源基因的两侧与核基质支架附着
区(matrix attachment region, MAR)连接, 能有效降
低位置效应, 消除基因沉默的影响, 在转基因可可
(Theobroma cacao)(Maximova et al., 2003)和烟草
(Halweg et al., 2005)等中均有成功的报道, 在转基
因杨树中也获得了成功(Han et al., 1997)。
(8) 研究林木基因调控网络。基因表达相互影响、
相互制约的关系构成了复杂的基因表达调控网络。林
木某些单个基因在不同时空条件下可参与不同的代
谢途径。例如美洲山杨的MADS-box开花基因PTM5
同时参与根维管组织的发育和根生物量的积累
(Cseke et al., 2007)。欧洲赤松(Pinus sylvestris)
PsRLK具有抗病基因LRR-XI的相似结构域, 其转录
物集中在韧皮部表达 , 可能参与木材发育的调控
(Avila et al., 2006)。喜树的CaAOC基因受盐胁迫和
低温的诱导, 研究表明, 其同时具备增强植株耐盐性
和抗寒性的能力(Pi et al., 2009)。因此不能孤立地研
究单个基因及其表达规律, 我们需要运用系统生物学
的方法, 对林木不同组织部位、不同代谢途径的全部
基因的表达关系进行整体的分析和研究, 构建林木基
因调控网络模型, 在系统的框架下深入了解林木生物
学特性。在人类、拟南芥和水稻(Oryza sativa)等物种
中已开展了基因调控网络研究, 但在林木中还未见报
道。
一直以来林木基因工程研究中的基因绝大多数
从微生物或农作物引用而来, 数量极为有限。外源基
因与林木基因组的整合可能存在亲和性的问题, 其在
林木基因组中的整合方式、整合位点和插入拷贝数均
不明确, 容易被植物基因组的重组和修复系统识别,
引起转基因的重排, 发生DNA的缺失、倒位、易位、
重组等异常变化, 且有可能产生转基因沉默现象, 直
接干扰了外源基因功能的发挥 (王关林和方宏筠 ,
2002; 苏晓华等, 2009)。林木遗传资源丰富, 从林木
自身克隆的基因有利于实现基因的有效转化, 抑制重
组效应的发生, 并提高遗传稳定性。另外, 外源基因
导入引发的安全性问题也备受关注, 因此自身基因的
应用能够避免外源基因导入后可能产生的毒素或致
敏原, 从而消除转基因林木对环境及人类的潜在危
害, 增加转基因林木的生物安全性。随着克隆技术的
不断完善和林木基因克隆研究的不断深入, 必将加速
多年生林木的遗传改良进程, 促进不同育种目标的转
基因林木新品种的培育, 推动林木功能基因组学的研
李少锋等: 林木基因克隆研究进展 93
究进程。
致谢 感谢中国林业科学研究院林业研究所卢孟柱
研究员对本文提出的宝贵意见!
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Research Progress in Gene Cloning in Forest Trees
Shaofeng Li, Xiaohua Su*, Bingyu Zhang
Key Laboratory of Tree Breeding and Cultivation, State Forestry Administration, Research Institute of Forestry, Chinese
Academy of Forestry, Beijing 100091, China
Abstract Forest trees have rich germplasm resources and a wide array of genetic differences among germplasms.
Cloning and transforming genes that control important traits in tree species can be valuable in cultivating new clones with
excellent quality. However, many of the genes with potential application have not been discovered or isolated. In recent
years, a large number of genes related to important traits in forest trees have been identified and isolated with the estab-
lishment of forest cDNA libraries, the use of large-scale random expressed sequence tag sequencing, improvements in
cloning technology, and more specifically, the complete genome sequencing of Populus trichocarpa Torr. & Gray. This
information has laid a solid foundation for using transgenic technology to cultivate new varieties of forest trees for high
yield, fine quality, high stress tolerance and pest resistance. In this review, we summarize the progress in gene cloning of
forest trees over the last 20 years. We discuss some problems in gene cloning and application, as well as future applica-
tions and prospects for gene cloning and transgenic technology of forest trees.
Key words forest trees, gene cloning, research progress, transgenic technology
Li SF, Su XH, Zhang BY (2011). Research progress in gene cloning in forest trees. Chin Bull Bot 46, 79–107.
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* Author for correspondence. E-mail: suxh@caf.ac.cn
(责任编辑: 刘慧君)