Alternative splicing of flowering regulatory gene LFY in Arabidopsis thaliana

为研究拟南芥成花调控基因LFY,我们采用RT-PCR方法分离克隆了三种选择性剪接的片段,分别命名为LFY1239,LFY1263和LFY1275.序列分析表明LFY1263包含一个大小为1 263 bp的开放阅读框,与之前报道的LFY基因片段大小相同,而LFY1239在第一外显子的3'端缺失了36 bp,LFY1275在第一内含子的3'末端插入了12 bp.对几种片段表达部位的分析显示,LFY1239只能在营养生长期的莲座叶中表达,而LFY1263和LFY1275在营养生长期和花期的花器官和莲座叶中都可以检测到,并且,LFY1263呈现出主导地位,LFY1275与LFY1263表达的比例表现为花器官高于莲座叶,该比例的变化可能预示着与成花调控有关.     

荷花LEAFY基因的克隆及表达分析

LEAFY（LFY）基因是花分生组织形成的必需基因,在成花过程中发挥重要作用。本研究以荷花品种‘红霞满天’为材料,利用已经获得的荷花LFY基因片段,采用RT-PCR和RACE技术克隆得到荷花LFY基因cDNA序列,命名为NnLFY。NnLFY全长cDNA为1 494 bp,开放阅读框（ORF）为1 173 bp,编码390个氨基酸,预测其相对分子量为44 517.1 Da。与其他物种已知的LFY基因序列进行同源性对比分析并构建系统进化树,结果显示荷花LFY基因与甜瓜LFY基因的亲缘关系最近。实时荧光定量PCR结果表明,LFY基因在荷花幼叶期、花蕾期、盛花期的根、茎、叶、花中均有表达,其中,花蕾期各器官的表达量较高。     

黄瓜离体子叶节花芽和营养芽分化中CFL基因的表达

CFL基因是从黄瓜中克隆到的拟南芥LEAFY(LFY)同源基因.以离体黄瓜子叶培养物成花为实验体系,利用mRNA原位杂交技术对CFL基因在花芽和营养芽分化过程中的时空表达进行了分析.结果如下:在花芽分化过程中,CFL基因在花原基形成、花器官原基分化及各轮花器官形成之初强表达,在花器官形成以后表达减弱或不表达;在营养芽分化过程中,CFL基因在分生组织、叶原基和幼叶中有明显表达,在成熟组织中不表达.结果说明CFL基因的表达在黄瓜子叶节花芽和营养芽分化中原基的分化形成是必需的.结果提示CFL基因可能参与细胞分裂调控和启动、营养性分生组织向花分生组织转变等过程.     

LEAFY同源基因研究进展

LEAFY(LFY)同源基因存在于所有的陆生植物中,在植物花发育早期表达,并在花发育过程中抑制茎端分生组织的营养生长,调控花分生组织和花器官的形成,使转LFY基因植株提前开花,LFY同源基因与其上下游基因共同调控花发育过程.LFY同源基因的蛋白质结构在不同物种间保守性很高,但它们的表达部位差异很大.该文总结了近年来国内外已经克隆到的LFY同源基因的表达、功能及其在果树、花卉、粮食作物上的应用,以期为植物花发育的深入研究提供参考.     

苦瓜中与AGAMOUS相似基因启动子的克隆和表达载体的构建

McAG2基因是苦瓜中分离得到的与AGAMOUS相似基因,在花器官和果实中特异表达,参与花的第三轮和第四轮器官的形成.文章采用染色体DNA步移技术克隆得到长为1 417 bp的McAG2基因5'上游片段.序列分析显示此片段含有典型的TATA-box、CAAT-box、丰富的激素应答调控元件.为了研究这些调控元件,还构建了McAG2基因5'侧翼缺失和内含子缺失表达载体.     

西瓜果实特异启动子WSP功能区域的初步定位

西瓜AGPase的大亚基基因wml1的 5′端上游 15 73bp序列 ,是一个果实特异性启动子 (命名为WSP)。根据WSP内部酶切位点 ,获得了 3个不同 5′端缺失的启动子片段 (长分别为 12 0 1bp、898bp、795bp) ,并构建成植物瞬间表达载体 ,与含WSP的瞬间表达载体一起用基因枪的方法转入西瓜叶、茎、花及不同发育期果实中。瞬时表达结果表明 ,15 73bp、12 0 1bp、898bp的片段均能指导GUS基因在西瓜果实和花中特异性表达 ,但是表达强度和表达时期有所不同 ,795bp的片段不能指导GUS基因表达。推测在 180bp 5 5 1bp之间可能存在促进外源基因在果实发育后期表达的顺式作用元件 ,而果实特异调控区域可能位于 85 4bp 95 7bp之间。     

西瓜果实特异启动子WSP功能区域的初步定位

西瓜AGPase 的大亚基基因wml1的 5′端上游1573bp序列,是一个果实特异性启动子(命名为WSP)。根据WSP内部酶切位点,获得了3个不同5′端缺失的启动子片段（长分别为 1201bp、898bp、795bp）,并构建成植物瞬间表达载体,与含WSP的瞬间表达载体一起用基因枪的方法转入西瓜叶、茎、花及不同发育期果实中。瞬时表达结果表明,1573bp 、1201bp 、898bp的片段均能指导GUS基因在西瓜果实和花中特异性表达,但是表达强度和表达时期有所不同,795bp的片段不能指导GUS基因表达。推测在180bp-551bp之间可能存在促进外源基因在果实发育后期表达的顺式作用元件,而果实特异调控区域可能位于854bp-957bp之间。     

日本晚樱PrseSHP基因的克隆与功能分析

采用同源克隆方法结合RACE技术,从日本晚樱(Prunus lannesiana)品种‘大岛樱’中克隆到花发育调控相关的PrseSHP基因(GenBank登录号为GU362645)。PrseSHP基因序列全长1 223bp,包含1个长741bp的完整开放阅读框,编码246个氨基酸和1个终止密码子。分子系统发生分析表明,PrseSHP属MADS-box转录因子的PLE/SHP进化系,并与蔷薇科植物的SHP同源蛋白聚于同一进化分支;蛋白序列比对显示,该转录因子拥有M、I、K和C共4个结构域,且其C末端结构域中包含高度保守的AGⅠ和Ⅱ基序。基因表达分析表明,PrseSHP基因主要在‘大岛樱’的花瓣、雄蕊、雌蕊和幼果等器官中表达,在花萼中仅能检测到微弱的转录信号,在幼叶中不表达,与其他植物SHP同源基因的表达模式有一定的差别。功能分析显示,转PrseSHP基因拟南芥植株明显比野生型拟南芥弱小,转基因拟南芥在6~8片莲座叶后即抽薹开花,时间较野生型拟南芥(14~17片莲座叶后抽薹开花)明显提前,证明异位表达的PrseSHP基因能促进拟南芥早花,其在花发育过程中可能参与调控植物开花。     

朵丽蝶兰MADS-box基因DtpsMADS1的克隆与表达特性

植物MADS-box基因家族编码高度保守的转录因子,参与了包括花器官发育和开花在内的多种发育进程。为阐释兰科植物成花的分子调控机制,根据MADS-box基因保守序列设计简并引物,用RACE方法从朵丽蝶兰花葶中克隆到1个MADS-box家族基因,该基因cDNA全长960 bp,包含37 bp 5'UTR,一个738 bp的开放阅读框(ORF)和185 bp 3'UTR,共编码245个氨基酸。序列和系统进化树分析表明,该基因与其他植物的MADS-box基因具有很高的同源性,属于AP1/FUL-like亚家族,命名为DtpsMADS1,GeneBank登录号为JQ065097。实时荧光定量PCR检测结果显示:DtpsMADS1具有明显的组织表达特异性;在根和叶中,DtpsMADS1在花前期和花后期表达量较高;苗期和盛花期表达量较低;DtpsMADS1在花葶中的表达趋势与根和叶相似;而在花器官中,DtpsMADS1只有痕量表达。由此推断,DtpsMADS1可能参与开花进程调控,而不参与花器官的形态建成。     

朵丽蝶兰 MADS-box 基因 DtpsMADS1 的克隆与表达特性简

植物MADS-box基因家族编码高度保守的转录因子,参与了包括花器官发育和开花在内的多种发育进程。为阐释兰科植物成花的分子调控机制,根据MADS-box基因保守序列设计简并引物,用RACE方法从朵丽蝶兰花葶中克隆到1个MADS-box家族基因,该基因cDNA全长960 bp,包含37 bp 5′UTR,一个738 bp的开放阅读框(ORF)和185 bp 3′UTR,共编码245个氨基酸。序列和系统进化树分析表明,该基因与其他植物的MADS-box基因具有很高的同源性,属于AP1/FUL-like亚家族,命名为DtpsMADS1,GeneBank登录号为JQ065097。实时荧光定量PCR检测结果显示：DtpsMADS1具有明显的组织表达特异性;在根和叶中,DtpsMADS1在花前期和花后期表达量较高;苗期和盛花期表达量较低;DtpsMADS1在花葶中的表达趋势与根和叶相似;而在花器官中,DtpsMADS1只有痕量表达。由此推断,DtpsMADS1可能参与开花进程调控,而不参与花器官的形态建成。     

菲油果LEAFY基因的表达模式及启动子克隆

LEAFY(简称LFY)是植物花分生组织特征基因,在植物由营养生长向生殖生长转变过程中起着重要作用,是启动开花的枢纽。菲油果是一种新兴的果树资源,为研究菲油果LFY基因(FsLFY)的表达调控规律,本研究通过实时荧光定量PCR技术研究了FsLFY基因的时空表达模式,并通过染色体步移技术克隆了该基因的启动子序列。荧光定量PCR结果表明,FsLFY基因在菲油果花蕾不同发育阶段以及其他组织器官中均有表达。在花蕾中,小蕾期最高,中蕾期最低;组织器官中,营养枝茎段最高,花瓣最低。FsLFY基因启动子序列长度为2436 bp(GenBank登录号:KF766536),运用PLACE、PlantCARE等在线软件对其序列进行顺式作用元件分析,结果显示该序列不仅含有CAAT-box、TATA-box等核心启动子元件,而且还具有响应水分、光、赤霉素(GA)以及其他功能未知的顺式调控元件,表明FsLFY基因的表达受多种外界环境条件的调控。本研究为阐明菲油果的开花机理,以及通过分子育种手段使菲油果早花早果奠定了理论基础。     

An exploration of LEAFY expression in independent evolutionary origins of rosette flowering in Brassicaceae

Whereas most Brassicaceae produce flowers on an elongated inflorescence, a few lineages produce flowers directly from the vegetative rosette on elongated pedicels. Knowing the extent to which independent origins of rosette flowering involve the same developmental and genetic mechanisms could clarify the constraints acting on plant architectural evolution. Prior work in Idahoa, Ionopsidium, and Leavenworthia suggested that changes in the activity or expression of the flower meristem identity gene, LEAFY (LFY), played a role in all three origins of rosette flowering. Here we studied the developmental morphology of L. crassa and immunolocalization of LFY protein in Leavenworthia and Ionopsidium to further compare independent origins of rosette flowering. Leavenworthia crassa differs from Ionopsidium and Idahoa in producing ebracteate flowers. Flowers are, however, associated with "squamules," here interpreted as stipules of a cryptic bract. LFY was detected in L. crassa flower primordia but not in inflorescence meristems. In contrast, the rosette flowering Io. acaule accumulated LFY protein in the inflorescence meristem, whereas its inflorescence-flowering close relative, Io. prolongoi, did not. Thus, although different cases of rosette flowering likely entailed modifications of the same meristem identity program, distinct developmental genetic mechanisms appear to be involved in each case.     

拟南芥LEAFY基因在花发育中的网络调控及其生物学功能

重点综述了拟南芥花分生组织特征基因——LEAFY（LFY）基因及其同源基因在花发育中的网络调控及其生物学功能。LFY基因广泛表达于高等植物的营养性和生殖性组织。LFY基因需要与其他基因相互作用,並且表达量达到一定水平时才能促进成花。LFY基因处于成花调控网络的关键位置,不仅调控开花时间和花转变,而且在花序和花的发育中也起重要作用。碳源、植物激素等因子直接或间接地影响LFY基因的表达和作用。提示通过掌握LFY基因的表达调控规律进一步探讨成花机理的可行性。 Abstract:Recent research progress on regulation network and biological roles of LFY gene in Arabidopsis thaliana and its homologue genes in floral development are reviewed emphatically in the present paper.LFY gene expresses widely in both vegetative and reproductive tissues in different higher plants,therefore investigation on role of LFY gene on flowering is of general significance.LFY gene plays an important role to promote flower formation by interaction and coordination with other genes,such as TFL,EMF,AP1,AP2,CAL,FWA,FT,AP3,PI,AG,UFO,CO,LD,GA1 etc,and a critical level of LFY expression is essential.LFY gene not only controls flowering-time and floral transition,but also plays an important role in inflorescence and floral organ development.It was situated at the central site in gene network of flowering regulation,positively or negatively regulates the level or activities of flowering-related genes.Some physiological factors,such as carbon sources,phytohormones,affect directly or indirectly the expression and actions of LFY gene.This indicates that level of LFY expression can also be regulated with physiological methods.It is probable that we can explain the principal mechanism of flowering by regulation network of LFY gene.     

FALSIFLORA, the tomato orthologue of FLORICAULA and LEAFY, controls flowering time and floral meristem identity

Characterization of the tomato falsiflora mutant shows that fa mutation mainly alters the development of the inflorescence resulting in the replacement of flowers by secondary shoots, but also produces a late-flowering phenotype with an increased number of leaves below first and successive inflorescences. This pattern suggests that the FALSIFLORA (FA) locus regulates both floral meristem identity and flowering time in tomato in a similar way to the floral identity genes FLORICAULA (FLO) of Antirrhinum and LEAFY (LFY) of Arabidopsis. To analyse whether the fa phenotype is the result of a mutation in the tomato FLO/LFY gene, we have cloned and analysed the tomato FLO/LFY homologue (TOFL) in both wild-type and fa plants following a candidate gene strategy. The wild-type gene is predicted to encode a protein sharing 90% identity with NFL1 and ALF, the FLO/LFY-like proteins in Nicotiana and Petunia, and about 80 and 70% identity with either FLO or LFY. In the fa mutant, however, the gene showed a 16 bp deletion that results in a frameshift mutation and in a truncated protein. The co-segregation of this deletion with the fa phenotype in a total of 240 F2 plants analysed supports the idea that FA is the tomato orthologue to FLO and LFY. The gene is expressed in both vegetative and floral meristems, in leaf primordia and leaves, and in the four floral organs. The function of this gene in comparison with other FLO/LFY orthologues is analysed in tomato, a plant with a sympodial growth habit and a cymose inflorescence development.     

CnFL,a FLORICAULA/LEAFY homolog in Chrysanthemum nankingense is dramatically upregulated in induced shoot apical meristems

A FLORICOULA/LEAFY (FLO/LFY) homolog CnFL gene was isolated from the flower bud of a short-day Chrysanthemum nankingense plant during the flowering induction period. The sequence of CnFL contained a 1236 bp open reading frame that encoded a putative protein of 412 amino acids, which shared 68.67% homology with FLO and 60.23% homology with LFY. The spatial expression patterns of CnFL were analyzed using quantitative real-time PCR in different tissues and the apical meristem during short-day flowering induction. The results indicated that CnFL was highly expressed in the flower buds while its expression was also detected in the stems, young leaves, and vegetative apical meristem. During the period of flowering induction, CnFL expression increased remarkably and reached its highest levels after 15 days of induction. The expression of CnFL in the apical shoot after short-day flowering induction indicates that CnFL regulation is controlled primarily by photoperiodicity.