兰科(Orichidaceae)植物统称兰花，是开花植物中第二大科。兰花的花色变化非常丰富，是研究花着色机理的理想对象(朱根发和郭振飞, 2004)。云南省有丰富的野生兰花资源，共有兰科植物135属、764种和16个变种以及众多变异类型，是我国乃至世界的兰花资源宝库(关文灵等, 2006)。本文总结了兰科植物花色研究进展，主要包括花色素组成及其合成与调控的分子机理。

1兰科植物花色素组成
植物的花色通常是由花青素苷(anthocyainins)和/或类胡萝卜素(carotenoids)的组合而决定的。兰科植物花色表现出同样的趋势，黄色到橙色由类胡萝卜素决定，橙色到红色由花青素苷和类胡萝卜素组合决定，红色到紫色由花青素苷决定(Thammasiri et al., 1986; Matsui and Nakamura, 1988; Matsui, 1994; Tatsuzawa et al., 2010)。文心兰(Oncidium Gower Ramsey)的花瓣和萼片(黄色带有红色斑点)同时含有花青素苷和类胡萝卜素，类胡萝卜素主要是堇菜黄质(violaxanthin)的全反和9-顺异构体，花青素苷主要是矢车菊素(cyanidin)及其甲基衍生物芍药花素(peonidin) (Hieber et al., 2006)。其它兰科植物的花青素苷也主要是矢车菊素，其次是天竺葵素(pelargonidin)或芍药花素(Kuehnle et al., 1997;Fossen and Øvstedal, 2003; Tatsuzawa et al., 2010)。植物花色素的生物合成途径与分子调节机理已比较清楚(Tanaka et al., 2008)，为研究兰科植物花色变异提供了可能。

2兰科植物花青素苷的生物合成与分子调控机理
花青素苷属于类黄酮(flavonoids)化合物，是决定植物花色的最常见色素，贡献颜色范围从黄色到橙色到红色乃至紫色。花青素苷的生物合成途径主要受到结构基因、转录因子和环境条件的调控。一些兰科植物参与花青素苷生物合成的酶的基因已经得到克隆，并对其表达与花色关系有所了解，但已有研究主要集中于蝴蝶兰和文心兰，兰科其它种类则很少涉及。

2.1兰科植物花青素苷生物合成中的结构基因
兰科植物的查耳酮合成酶(CHS)基因由一个小的多基因家族编码，其在花器官的各部位和红色的幼叶均有表达，萼片表达水平最高，花尚未着色的发育初期表达水平最高(Mudalige-Jayawickrama et al., 2005; Liew et al., 1998a; Han et al., 2005; Pitakdantham et al., 2010; Han et al., 2006)。

二氢黄酮醇4-还原酶(DFR)是将二氢黄酮醇转变为花色素的第一个酶，在不同花色形成中起着关键作用，不同物种DFR对底物选择性不同，合成不同的花色素，呈现各异的花色。石斛兰(Dendrobium)缺乏橙色与蓝色，这与石斛兰花色形成过程中DFR的活性密切相关。兰科植物DFR基因多数为单拷贝(Liew et al., 1998b)，少数为多拷贝(Hieber et al., 2006)。兰属植物DFR不能有效还原DHK(二氢山奈酚, dihydrokaempferol)，导致天竺葵素缺乏(Johnson et al., 1999)而使其缺乏橙色，这可能是由于DFR和类黄酮-3’-羟化酶(F3′H)竞争相同底物DHK的结果(Mudalige-Jayawickrama et al., 2005)。

蓝色和紫色花通常含有飞燕草素(delphinidin)，类黄酮-3’,5’-羟化酶(F3’5’H)是生物合成飞燕草素的关键酶。蝴蝶兰F3’5’H基因的表达与花发育时期和花色密切相关，在完全开放的时期和紫色花中表达水平最高，而在黄色和白色花中水平很低，叶片和根中无表达(Wang et al., 2006)，蝴蝶兰F3’5’H基因瞬间表达于花瓣，其颜色由粉红转为洋红(Su and Hsu, 2003)。

UDP-葡萄糖：类黄酮3-O-糖基转移酶(UFGT)是形成稳定的花色素苷所必需的。红色和白色蝴蝶兰品种CHS，CHI(查耳酮异构酶基因)和ANS(花青素合成酶基因)表达水平没有差别，但UFGT基因仅在红花中有强烈表达，因此UFGT基因与红花形成高度相关(Chen et al., 2011)。

2.2转录因子对兰科植物花青素苷生物合成的调控
花青素苷生物合成途径中结构基因的时空表达由3种转录因子及其相互作用而决定，分别是R2R3-Myb，Myc基因家族的bHLH (basic helix-loop-helix)和WD40-型转录因子(Tanaka et al., 2008)。Wu(2003)等最早从石斛属(Dendrobium)中分离出21个R2R3-Myb基因(Wu et al., 2003)。

蝴蝶兰(Phalaenopsis amabilis)白色花瓣中同时表达玉米(Zea mays) Myc Lc和Myb C1可激活花青素苷的合成，而单独表达其中任何一个则不能激活，说明其花青素苷的生物合成需要上述两种转录因子(Griesbach and Klein, 1993)，这种激活作用也与植物基因启动子有关(Ma and Pooler, 2008)。白花蝴蝶兰(P. amabilis) DFR表达水平很低，紫花蝴蝶兰(P. schilieriana) DFR表达水平很高，二者均表达Myc和Wd基因，但Myb基因只表达在后者中有表达，说明缺乏Myb基因使白花蝴蝶兰丧失DFR活性，致使不能合成花青素苷(Ma and Pooler, 2009)。在文心兰的黄色唇瓣中瞬间表达OgMYB1，可以通过激活CHI和DPR基因的转录，而诱导产生红色(Chiou and Yeh, 2008).最新的研究表明，玉米花青素转录因子Leaf color，Colorless 1和金鱼草(Anthurium andraeanum) AaMYB1均可以激活墨兰(Cymbidium)花青素苷的合成(Albert et al., 2010)。

2.3环境因子对植物花青素苷合成的调控
环境因子调控花青素苷生物合成途径中的结构基因与转录因子的表达，从而影响花青素苷种类与含量(胡可等, 2010)。影响花青素苷合成的环境因子包括光照、温度、糖、水份、氮或磷等(Weiss, et al., 2000; Rajendran et al., 1992)。光照强度和光质均影响花青素苷的合成和植物花、果实和叶片等的呈色。光信号通过激活花青素苷合成途径上相关基因的表达直接或间接地调控花青素苷的合成与积累(唐杏姣和戴思兰, 2011)。强光促进结构基因和调节基因的表达，促使花青素苷积累；而黑暗或弱光下调基因的表达，产生白花或浅色花(胡可等, 2010)，如拟南芥(Arabidopsis thaliana)、矮牵牛(Petunia hybrida)、葡萄(Vitis vinfera)和百合(Lilum spp.)等(Cominelli et al., 2008; Albert et al., 2009; Matus et al., 2009; Nakatsuka et al., 2009)。UV-B是大多数植物花花青素苷呈色所必需的(Dong et al., 1998)，UV-A和低强度的UV-B可以诱导花青素苷生物合成相关基因的表达，从而诱导花青素苷的积累(Guo et al., 2008)，科特兰(Cattleya)和惠兰(Cymbidium)叶片叶绿素、类胡萝卜素和类黄酮的含量均受到紫外辐射的影响(Zhao et al., 2003)。

温度升高时花青素苷合成速率减慢，降解速率加快导致其积累减少(Shaked-Sachray et al., 2002)，如长叶车前(Plantago lanceolata)，葡萄和菊花(Chrysanthemum morifolium)均是如此(Stiles et al., 2007; Mori et al., 2007; Poudel et al., 2009; Nozaki et al., 2006; Huh et al., 2008)。缺氮、缺磷或二者同时缺少导致花青素苷含量增加(Rajendran et al., 1992; Lillo et al., 2008)。外源NH4+抑制非洲菊(Gerbera hybrida)花瓣色素的积累(Huang et al., 2008),而外源腐胺能够有效促进苹果花青素苷的积累(田义等, 2009)。综上所述，花青素苷的生物合成受到多种环境因子的调控，但哪个起主导作用及其作用途径尚不清楚。同时，相关研究主要集中于拟南芥和矮牵牛等少数模式植物上，环境因子对兰科植物花色的影响和调控机理等尚未有报道。

3兰科植物类胡萝卜素的生物合成与分子调控机理
类胡萝卜素是一类积累于非光合质体中的脂溶性色素，多数植物的主要是黄色的堇菜黄素(Tanaka et al., 2008)，包括兰科植物中的文心兰(Hieber et al., 2006; Chiou et al., 2010)。研究发现，黄花的文心兰品种Gower Ramsy积累堇菜黄素新黄质(neoxanthin)，橙花品种Sunist除二者外还含有β-胡萝卜素(β-carotene)，白花品种White Jade则不含类胡萝卜素类化合物(Hieber et al., 2006)。进一步的研究表明，由于橙花品种中β-羟化酶基因(β-hydroxylase, HYB)和玉米黄质环氧酶基因(zeaxanthin epoxidase, ZEP)转录水平低于其在红花品种的转录水平，导致堇菜黄质和新黄质含量减少，β-胡萝卜素含量增加，而表现出橙色；白花品种则是由于9-顺式-环氧类胡萝卜素双加氧酶(9-cis-epoxycarotenoid dioxygenase, NCED)基因和类胡萝卜素裂解双加氧酶(carotenoids cleavage dioxygenase, CCD)基因表达水平高，导致降解为无色的脱辅基类胡萝卜素(apocarotenoid) (Chiou et al., 2010)。类胡萝卜素生物合成途径中各酶及其编码基因的成功分离，为鉴定其关键的调节步骤提供了基础，但其分子调节机理仍然缺乏了解(Tanaka et al., 2008)，环境因子对类胡萝卜素的合成与呈色影响等也少有研究。最新的研究表明，一些光信号因子如HY5和COP1对番茄果实类胡萝卜素的生物合成有相反的调节作用(Liu et al., 2004; Davuluri et al., 2005)。

4问题与展望
兰科植物的花色素组成主要是花青素苷和类胡萝卜素。近年来花青素苷的生物合成与调控分子机理研究很多，对一些环境因子如光的调控机理等也了解比较深入(唐杏姣和戴思兰, 2011)，但是研究多数集中于模式植物，兰科植物涉及较少，特别是国兰的种类。云南有丰富的兰花资源和多变的地理环境，一些稀有的兰花种类，如莲瓣兰(Cymbidium Lianpan)等其花色很少有报道。课题组在收集野生资源过程中发现，兰花生长地的环境条件，如海拔、紫外辐射、温度等对其花色有显著影响，这些环境因子也是形成云南兰花变异众多的原因。因为云南特有兰花资源幼苗稀少、价格高昂，对研究工作造成很大的影响，同时一些环境因子如海拔高度对花色变异的影响因为很难创造模拟条件，研究难度很大。因此，必需首先将云南特有兰花资源进行花色初步分类，然后重点研究各类花色组成和调控分子机理，特别转录因子对花色的调控以及环境条件对转录因子表达的影响。

同时，课题组在研究中也发现，多数兰科植物缺乏红色、蓝色和紫色。兰花缺乏红色系和紫色系是结构基因和转录因子共同作用的结果。前人的研究表明，瞬间表达花青素苷生物合成途径的转录因子，能够促进兰花花色改变而产生红色(Griesbach and Klein, 1993; Chiou and Yeh, 2008; Ma and Pooler, 2009;Albert et al., 2010)，而且在模式作物上的研究也表明，超表达花青素苷转录因子不但能够改变植物花色(Lloyd et al., 1992)，而且能够改变营养器官如叶片的颜色(Golesbrough et al., 1996; Elomaa et al., 2003; Ray et al., 2003)。因此，超表达花青素苷转录因子是兰花花色育种的有效途径之一。

近年兰科植物的离体培养(Chugh et al., 2009)和遗传转化均取得较大进展，但依然落后于其他草本植物，多数研究仍集中于转化体系建立(Zhang et al., 2010; Shrestha et al., 2007; Semiarti et al., 2010; Sjahail and Mii, 2006)。壳聚糖(chitosan)能够促进石斛兰属原球茎(protocorn-like body)的增殖、芽分化和幼苗发育(Pornpienpakdee et al., 2010)，而且能够促进其种子萌发(Kananont et al., 2010)。最新研究发现，蝴蝶兰原球茎在添加番茄提取物的培养基上预培养，能够促进抗性芽的再生，转化频率高达10%~14% (Semiarti et al., 2011)。这些研究为兰花花色分子育种，特别是培育红色、蓝色和紫色兰花提供了可能。

作者贡献
王有国和张广辉完成文献检索和初稿的写作，孙慧晶和郭红参与了论文的写作和修改。李枝林审阅了全文。全体都阅读并同意最终的文本。                
                    
致谢 
本研究工作得到云南教育厅科学研究基金(项目编号: 09Y0195)资助。                                    

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