草原龙胆MADS-box基因的克隆及表达分析

植物MADS-box 基因家族编码高度保守的转录因子, 参与了包括花发育在内的多种发育进程。为阐释双子叶植物草原龙胆(Eustoma grandiflorum)花器官发育的分子调控机制, 根据MADS-box基因保守序列设计简并引物, 用3'-RACE方法从 草原龙胆中克隆了4个花器官特异表达的MADS-box家族基因。序列和系统进化树分析表明, 这4个基因分别与金鱼草DEF基因、矮牵牛FBP3基因和FBP6基因以及拟南芥SEP3基因具有很高的同源性, 分别属DEF/GLO、AG-like和SEP-l ike亚家族。从而将这4个基因分别命名为EgDEF1、EgGLO1、EgPLE1和EgSEP3-1。推导的氨基酸序列显示, 这些基因编码的蛋白质都包含高度保守的MADS结构域、I结构域和K结构域, 每个基因均有其亚家族特异的C-末端功能域。基因特异性RT-PCR检测结果显示: EgDEF1 在萼片、花瓣、雄蕊及胚珠中高丰度表达, 在心皮中微量表达; 而EgGLO1在花瓣和雄蕊中高丰度表达, 在萼片中微量表达; 在根、茎、叶等营养器官中均未检测到上述2个基因的表达。EgPLE1在雌蕊、心皮和胚珠中特异表达, 但表达的丰度存在差异, 在雄蕊中的表达有所减弱。SEP-like亚家族基因EgSEP3-1在四轮花器官和胚珠中均特异表达,且表达丰度相对一致。     

草原龙胆MADS-box基因的克隆及表达分析

植物MADS-box基因家族编码高度保守的转录因子,参与了包括花发育在内的多种发育进程。为阐释双子叶植物草原龙胆（Eustoma grandiflorum）花器官发育的分子调控机制,根据MADS-box基因保守序列设计简并引物,用3＇-RACE方法从草原龙胆中克隆了4个花器官特异表达的MADS-box家族基因。序列和系统进化树分析表明,这4个基因分别与金鱼草DEF基因、矮牵牛FBP3基因和FBP6基因以及拟南芥SEP3基因具有很高的同源性,分别属DEF/GLO、AG-like和SEP-like亚家族。从而将这4个基因分别命名为EgDEF1、EgGLO1、EgPLE1和EgSEP3-1。推导的氨基酸序列显示,这些基因编码的蛋白质都包含高度保守的MADS结构域、I结构域和K结构域,每个基因均有其亚家族特异的C-末端功能域。基因特异性RT-PCR检测结果显示：EgDEF1在萼片、花瓣、雄蕊及胚珠中高丰度表达,在心皮中微量表达;而EgGLO1在花瓣和雄蕊中高丰度表达,在萼片中微量表达;在根、茎、叶等营养器官中均未检测到上述2个基因的表达。EgPLE1在雌蕊、心皮和胚珠中特异表达,但表达的丰度存在差异,在雄蕊中的表达有所减弱。SEP-like亚家族基因EgSEP3-1在四轮花器官和胚珠中均特异表达,且表达丰度相对一致。     

黄金树B类MADS-box基因表达特征分析

采用同源克隆技术, 从黄金树(Catalpa speciosa)花芽中克隆得到B类MADS-box基因CaspAP3和CaspPI的cDNA序列。序列分析表明, CaspAP3基因cDNA序列的完整开放阅读框(ORF)为696 bp, 编码231个氨基酸残基; CaspPI基因cDNA序列的ORF为639 bp, 编码212个氨基酸残基。蛋白质序列相似性比对和分子系统发生分析表明, CaspAP3属于AP3/DEF进化支, 其C末端包含保守的euAP3基序和PI-derived基序, 而CaspPI聚类于PI/GLO进化支, 其C末端包含保守的PI基序。半定量RT-PCR分析结果表明, CaspAP3和CaspPI基因均仅在花瓣和雄蕊中表达。实时荧光定量PCR分析表明, CaspAP3和CaspPI基因在花瓣和雄蕊原基分化期至成熟期均有表达, 这2个基因在雄蕊中表达高峰出现的时间均早于花瓣; 且花瓣中的CaspAP3和CaspPI基因表达高峰均出现在快速伸长阶段; 这与花瓣和雄蕊的形态发育阶段相吻合。     

兜兰DEFICIENS(DEF)-和GLOBOSA(GLO)-like基因的克隆及表达分析

以‘同色兜兰’品种为材料,采用RT-PCR和RACE技术获得了DEFICIENS(DEF)-和GLOBOSA(GLO)-like基因的cDNA全长,命名为PcDEF和PcGLO,并用半定量RT-PCR和实时PCR研究了PcDEF和PcGLO在花芽发育过程和不同组织部位的表达特性。结果表明,PcDEF和PcGLO的全长cDNA分别为1 039bp和934bp,分别编码224和210个氨基酸;蛋白比对表明,PcDEF和PcGLO蛋白都具有典型MADS-box蛋白的MADS和K结构域;蛋白同源性分析显示,PcDEF和PcGLO与已登录的其它兰科植物的DEF/AP3和GLO/PI蛋白的相似性分别在75%～96%和87%～98%;系统进化树分析表明,PcDEF和PcGLO分别属于B类MADS-box蛋白家族的AP3和PI亚家族。表达分析显示,PcDEF和PcGLO在花芽发育中均有表达,PcDEF在成熟花、唇瓣和花瓣中的表达量高,在蕊柱、萼片、苞叶和根中次之,在花茎和叶中较低,在子房中几乎不表达;PcGLO在各组织中均有不同丰度的表达。     

拟南芥AtPI基因植物表达载体的构建及其在烟草中的遗传转化

花是被子植物主要的繁殖器官,在繁育后代的过程中,发挥着极其重要的作用,PISTILLATA(PI)基因作为控制花器官发育的B类功能基因中的一员,在花器官发育中起到重要的作用。为探究PI基因在花瓣和雄蕊发育中的功能,本文以拟南芥(Arabidopsis thaliana)中的PI基因作为研究对象,利用PCR技术从拟南芥花序c DNA扩增出At PI基因,构建植物表达载体(p ROKⅡ-At PI)并进行烟草(Nicotiana tobacum)转化。转基因植株的PCR检测结果表明,At PI基因已经整合到了烟草基因组中。在T2代植株中,通过实时定量荧光PCR检测显示,At PI在m RNA水平也均有表达。过量表达PI的转基因烟草在花器官中存在明显表型,与野生型相比主要表现为转基因植株花冠变小,雄蕊缩短,果实畸形且子房基部比野生型长5~10 mm,上述结果表明At PI基因是特异性参与雄蕊和花瓣的发育并起着至关重要的作用。     

Col生态型拟南芥AP3基因启动子克隆及植物表达载体构建

隶属于MADS-Box基因家族的拟南芥花器官B类特征基因APETALA3 (AP3)在花瓣和雄蕊中特异性地表达;AP3基因编码转录因子,与A类和C类特征基因协同作用控制双子叶植物花瓣和雄蕊的发育.研究表明AP3基因启动子为花特异表达启动子.因此,AP3基因启动子的克隆及功能鉴定对于园林植物与花相关的商业性状的定向改良具有重要作用.本文根据GenBank数据库报道的Ler生态型拟南芥(Arabido-psis thaliana) AP3基因启动子序列(U30729)设计了一对特异性扩增引物,基于PCR技术,用高保真的KOD-plus DNA聚合酶扩增了长度为1 767 bp的Col生态型拟南芥AP3基因启动子,并命名为pAtAP3,其GenBank登录号为FJ619533.Bl2seq在线分析表明pAtAP3与U30729序列的相似性达98%,与Col生态型拟南芥BAC克隆T12E18 (AL132971) 9 264～11 030之间的碱基序列相似性达100%,且该段序列的下游基因编码AP3蛋白(CAB81799),说明克隆序列为Col生态型拟南芥AP3基因的启动子.PLACE在线分析表明pAtAP3具有基本的启动子元件TATA-box和CAAT-box,还包含大量与花特异表达相关的顺式元件CArG1、CArG2、CArG3和anther-box等.本试验进一步构建了植物表达载体pAtAP3::GUS,为该启动子的功能鉴定奠定了基础.     

棉花MADS框基因GhMADS3的克隆及其组成型表达对烟草花器官决定的影响

MADS框基因在植物花器官发育中发挥着关键性作用。为研究棉花花器官发育的机理,以徐州142花蕾为材料,利用EST数据库资料,通过EST序列整合,克隆出了一个MADS域蛋白的编码区段,GenBank登录号为AY083173。该片段(GhMADS3)包含一个732 bp的开放阅读框,推导的氨基酸序列(244氨基酸)与可可,黄瓜,烟草,矮牵牛,金鱼草等的AG亚家族基因的序列相似性高。进化树重建分析将GhMADS3基因归入MADS框基因AG亚家族C功能分支的euAG分支。RT-PCR分析显示,该基因在雄蕊和心皮中表达,在根、茎、叶等营养器官,萼片,花瓣,花器官变异体chv1(所有花器官均变为苞叶状器官)的花蕾中不表达。将GhMADS3与35S启动子融合构建成嵌合基因转化烟草,转基因烟草植株花朵出现萼片(轮1)向心皮,花瓣(轮2)向雄蕊的转变,花器官表现明显的白化倾向。同时,在轮1观察到丝状结构的出现,该结构在此前类似的研究中尚无报道。这些结果说明,实验中克隆了一个有生物学功能的棉花的AG亚家族MADS框基因,该基因可能在棉花花器官发育中有重要的功能。     

蝴蝶兰PhalPI基因的克隆及在花器官突变体中的表达分析

为深入研究兰科植物花器官发育的调控机理,从蝴蝶兰花瓣中克隆了一个B类MADS-box转录因子PhalPI（GenBank登录号为KY020416）。序列分析表明,该基因的cDNA全长为944 bp,含完整的开放阅读框,可编码210个氨基酸,属于BGLO/PI蛋白家族,与蝴蝶兰属的PhPI10和PeMADS6基因关系最近;表达模式分析表明,PhalPI基因在生殖器官中表达,在营养器官中不表达,在授粉后的子房中,该基因的表达水平降低。在5种花器官突变体中,PhalPI基因在萼片唇瓣化突变体的萼片和蕊柱中表达水平明显升高;在雄蕊花瓣化突变体的萼片和侧瓣中表达水平降低,在其唇瓣和蕊柱中显著升高;在侧瓣合柱化突变体的蕊柱中,PhalPI基因的表达也发生了显著升高;PhalPI基因表达的改变与花器官形态的突变相关;而在侧瓣唇瓣化和侧瓣花药化突变体中,PhalPI基因的表达水平没有变化。推测该基因在决定蝴蝶兰侧瓣和唇瓣的发育中起重要的调控作用。     

大豆GmAP1基因的功能初探

以大豆天隆1号为材料,根据Glycine max Wm82.a2.v1中大豆基因组的预测序列,克隆出一个AP1同源基因,命名为GmAP1。该基因编码区CDS长度为711 bp,编码一个含236个氨基酸的蛋白质。对蛋白质序列的结构分析结果显示GmAP1蛋白符合MADS-box基因家族特征。为了初步分析GmAP1的功能,利用实时定量RT-PCR分析GmAP1在不同花器官中表达,并且在拟南芥中过表达该基因。在不同花器官中,GmAP1表达量不同,在花萼中表达量较高;花瓣次之。GmAP1过表达植株表现出早花、植株矮小以及花器官(花瓣、雄蕊和雌蕊)数量增多等表型。结果表明,大豆的GmAP1是一个功能保守的AP1基因,在花的发生和花器官发育中起着重要作用。     

无花瓣油菜雄蕊心皮化突变体细胞学观察及基因表达分析

油菜是我国重要的油料作物,油菜花器官具有典型的十字花科特点,无花瓣油菜在花期不存在花冠层,这种特点有助于提高油菜产量,预防茵核病的传播。雄蕊心皮化是指花器官的雄蕊结构被具有类似于雌蕊结构的器官代替,这不仅造成了花器官结构的变化也导致了雄性不育。本文通过对无花瓣油菜雄蕊心皮化突变不育分离群体中的雄性可育株和不育株的比较研究,发现心皮化现象是由遗传因素引起的。细胞学观察发现,雄蕊心皮化在花器官发育的早期就已经产生,心皮化的雄蕊中着生类似于胚珠的结构,其顶端细胞的形态和排列方式也与雌蕊柱头相似。花发育相关基因的表达分析表明,B组基因彳丹在不育株3轮花器中的表达都比可育株低,特别是在第二轮花器官中这种差异最为突出。而A组基因AP1在不育株第二轮花器官中的表达量较可育株高。c组基因AGL8、SHPI、SHP2、NAP在不育株心皮化的第二轮花器官中表达都较可育株中高。     

The duplicated B-class heterodimer model: whorl-specific effects and complex genetic interactions in Petunia hybrida flower development

In both Antirrhinum (Antirrhinum majus) and Arabidopsis (Arabidopsis thaliana), the floral B-function, which specifies petal and stamen development, is embedded in a heterodimer consisting of one DEFICIENS (DEF)/APETALA3 (AP3)-like and one GLOBOSA (GLO)/PISTILLATA (PI)-like MADS box protein. Here, we demonstrate that gene duplications in both the DEF/AP3 and GLO/PI lineages in Petunia hybrida (petunia) have led to a functional diversification of their respective members, which is reflected by partner specificity and whorl-specific functions among these proteins. Previously, it has been shown that mutations in PhDEF (formerly known as GREEN PETALS) only affect petal development. We have isolated insertion alleles for PhGLO1 (FLORAL BINDING PROTEIN1) and PhGLO2 (PETUNIA MADS BOX GENE2) and demonstrate unique and redundant properties of PhDEF, PhGLO1, and PhGLO2. Besides a full homeotic conversion of petals to sepals and of stamens to carpels as observed in phglo1 phglo2 and phdef phglo2 flowers, we found that gene dosage effects for several mutant combinations cause qualitative and quantitative changes in whorl 2 and 3 meristem fate, and we show that the PHDEF/PHGLO1 heterodimer controls the fusion of the stamen filaments with the petal tube. Nevertheless, when the activity of PhDEF, PhGLO1, and PhGLO2 are considered jointly, they basically appear to function as DEF/GLO does in Antirrhinum and to a lesser extent as AP3/PI in Arabidopsis. By contrast, our data suggest that the function of the fourth B-class MADS box member, the paleoAP3-type PETUNIA HYBRIDA TM6 (PhTM6) gene, differs significantly from the known euAP3-type DEF/AP3-like proteins; PhTM6 is mainly expressed in the developing stamens and ovary of wild-type flowers, whereas its expression level is upregulated in whorls 1 and 2 of an A-function floral mutant; PhTM6 is most likely not involved in petal development. The latter is consistent with the hypothesis that the evolutionary origin of the higher eudicot petal structure coincided with the appearance of the euAP3-type MADS box genes.     

To B or Not to B a flower: the role of DEFICIENS and GLOBOSA orthologs in the evolution of the angiosperms

DEFICIENS (DEF) and GLOBOSA (GLO) function in petal and stamen organ identity in Antirrhinum and are orthologs of APETALA3 and PISTILLATA in Arabidopsis. These genes are known as B-function genes for their role in the ABC genetic model of floral organ identity. Phylogenetic analyses show that DEF and GLO are closely related paralogs, having originated from a gene duplication event after the separation of the lineages leading to the extant gymnosperms and the extant angiosperms. Several additional gene duplications followed, providing multiple potential opportunities for functional divergence. In most angiosperms studied to date, genes in the DEF/GLO MADS-box subfamily are expressed in the petals and stamens during flower development. However, in some angiosperms, the expression of DEF and GLO orthologs are occasionally observed in the first and fourth whorls of flowers or in nonfloral organs, where their function is unknown. In this article we review what is known about function, phylogeny, and expression in the DEF/GLO subfamily to examine their evolution in the angiosperms. Our analyses demonstrate that although the primary role of the DEF/GLO subfamily appears to be in specifying the stamens and inner perianth, several examples of potential sub- and neofunctionalization are observed.     

A DEF/GLO-like MADS-box gene from a gymnosperm: Pinus radiata contains an ortholog of angiosperm B class floral homeotic genes.

The specification of floral organ identity during development depends on the function of a limited number of homeotic genes grouped into three classes: A, B, and C. Pairs of paralogous B class genes, such as DEF and GLO in Antirrhinum, and AP3 and PI in Arabidopsis, are required for establishing petal and stamen identity. To gain a better understanding of the evolutionary origin of petals and stamens, we have looked for orthologs of B class genes in conifers. Here we report cDNA cloning of PrDGL (Pinus radiata DEF/GLO-like gene) from radiata pine. We provide phylogenetic evidence that PrDGL is closely related to both DEF- and GLO-like genes of angiosperms, and is thus among the first putative orthologs of floral homeotic B function genes ever reported from a gymnosperm. Expression of PrDGL is restricted to the pollen strobili (male cones) and was not detected in female cones. PrDGL expression was first detected in emergent male cone primordia and persisted through the early stages of pollen cone bud differentiation. Based on the results of our phylogeny reconstructions and expression studies, we suggest that PrDGL could play a role in distinguishing between male (where expression is on) and female reproductive structures (where expression is off) in radiata pine. We speculate that this could be the general function of DEF/GLO-like genes in gymnosperms that may have been recruited for the distinction between stamens and carpels, the male and female reproductive organs of flowering plants, during the evolution of angiosperms out of gymnosperm-like ancestors.