【作者】 徐启江；

【导师】 李玉花；

【作者基本信息】 东北林业大学 ， 森林生物工程， 2007， 博士

【摘要】 草原龙胆(Eustoma grandiflorum)为龙胆科(Gentianaceae)龙胆属(gentian)观赏植物,原产于美国中南部,因其适应性强、花姿典雅、花茎颀长、花色丰富艳丽而成为国际流行的鲜切花。草原龙胆既具有单瓣花又有重瓣花,重瓣花的四轮花器官发育正常且雄蕊数目稳定,这与重瓣花多存在雄蕊瓣化的现象不同。为了阐释草原龙胆花器官发育的分子机理,探讨花器官特征MADS-box基因的保守性及表达情况,本研究以用草原龙胆黄色单瓣(49号)及重瓣花(50号)品系为试验材料,通过抑制性消减杂交及3’-RACE技术,克隆了草原龙胆的DEF/AP3亚家族(B功能基因)、GLO/PI亚家族(B功能基因)、PLE/AG亚家族(C功能基因)和SEP3/AGL9亚家族(E功能基因)MADS-box基因,开展了序列分析、系统发生树构建和RT-PCR等试验工作,为深入研究草原龙胆花发育的分子机理提供有用信息。1利用SSH技术筛选草原龙胆花器官发育特征基因以草原龙胆(Eustoma grandiflorum)不同发育时期的花器官(萼片,花瓣,雄蕊,雌蕊)原基的cDNA作为试验方(tester),以茎叶组织的cDNA作为驱动方(driver),利用抑制性消减杂交(suppression subtractive hybridization,SSH)技术构建了一个富集花器官发育特性基因的抑制性差减cDNA文库。对抑制性差减cDNA文库进行筛选、测序,Blast同源性比较。对抑制性差减cDNA文库进行筛选、测序,Blast同源性比较结果表明:共分离出1610条长度在200 bp以上的序列(平均长度341 bp,最长片段696 bp)。去除冗余序列,经过SeqClust2.1软件拼接得到非重复序列667条,与GenBank数据具有同源性的330条,占49.5%;没有显著同源性的337条序列,占50.5%。在有同源性的序列中,功能已知的有310条,功能未知的20条。序列分析结果表明,部分序列与金鱼草的DEF基因、冬青(Ilex aquifolium)的AP3基因、葡萄(Vitis vinifera)的MADS3基因、矮牵牛(Petunia hybrida)的FBP3基因、灌状买麻藤(Gnetum gnemon)的GGM6基因具高度同源性。成功构建了抑制性差减cDNA文库,为克隆草原龙胆花器官特性基因奠定了基础。2草原龙胆MADS-box基因克隆及系统进化分析以草原龙胆花芽及成熟的花器官为试验材料,提取RNA,根据MADS-box基因保守序列设计兼并引物,用3’-RACE方法从草原龙胆中分离33条花器官MADS-box基因的cDNA片段,并克隆了5个花特异表达的MADS-box家族基因,序列和系统进化树分析表明这5个基因分别与金鱼草的DEF基因、矮牵牛FBP3基因、FBP6基因和FBP7基因以及与拟南芥的SEP3基因具有很高的同源性,分属DEF/GLO亚家族(B功能基因)、AG-like亚家族(C/D功能基因)和SEP-like亚家族(E功能基因),从而将这5个基因分别命名为EgDEF1、EgGLO1、EgPLE1、EMADS1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,每一基因都有其亚家族特异的C-末端功能域。3草原龙胆MADS-box基因的表达分析为了分析草原龙胆花发育相关MADS-box基因在野生型花器官中的表达空间特异性,以草原龙胆的根、茎、叶、萼片、花瓣、雄蕊、雌蕊和胚珠等器官的总RNA为模板,进行基因特异性RT-PCR检测。结果显示:B功能MADS-box基因EgDEF1在萼片、花瓣、雄蕊及胚珠中有高丰度表达,在心皮中有微量表达;而EgGLO1在花瓣、雄蕊中有高丰度表达,在萼片中有微量表达;在根、茎、叶营养器官中均未检测到两个基因的表达。C功能基因EgPLE1在雄蕊、心皮和胚珠中特异表达,但表达的丰度存在一定的差异,在雄蕊中的表达有所减弱。D功能基因EgMADS1在心皮和胚珠中特异高丰度表达。E功能基因EgSEP3-1在四轮花器官和胚珠中均特异表达,且表达的丰度相对一致。更多还原

【Abstract】 Eustoma grandiflorum Grise.is a gentian native to the central and southern United States. Due to its large flowers,long stems,a wide range of flower colors and extended vase life,its cut flowers become increasingly popular.There are cultivars with simple and double flowers. In double flowers,the developments of four whorl organs are normal and the number of stamens is stable.In order to elucidate the molecular mechanism of floral development, investeigation was made on the conservation and divergence of expression patterns of floral organ identity MADS-box genes.Several MADS-box genes,including DEF/AP3 subfamily(B class),GLO/PI subfamily(B class),PLE/AG subfamily(C class),and SEP3/AGL9 subfamily (E class),were isolated from Eustoma grandiflorum,yellow simple(No.49) and double flowers(No.50) lines,by SSH(suppression subtractive hybridization) and 3’-RACE(Rapid amplification of cDNA ends).Sequence analyses,phylogeny reconstruction and RT-PCR expression analyses were performed.The gene structure and expression obtained in this investigation can provide important information regarding the floral development of Eustoma grandiflorum.1.Primary screening of floral organ identity genes during floral development with suppression subtractive hybridization in Eustoma grandiflorumThe purpose of this study was to get floral identity genes from Eustoma grandiflorum using SSH.A subtractive cDNA library,which was enriched in genes related to flower organ characteristics,was constructed.The cDNA prepared from sepal,petal,androecium,and gynoecium at different developmental stages was used as the tester and the cDNA from stem and leaf organ as the driver.From the library,1610 high quality sequences(longer than 200 bp, 696 bp at the longest and 341 bp in average) were isolated.After clustering,these unigenes were contigged by SeqClust 2.1 software,and then we got 667 contig sequences.By running blastn program of NCBI,330 contig sequences were found to have homologous genes with the network database,and 337 ESTs(expressed sequence tags) without remarkable homology. Among 330 ESTs with homology,there were 310 ESTs with known function and 20 ESTs with unknown function.The results of GenBank search indicated that they showed a high degree of identity in nucleotide sequence with DEF gene from Antirrhinum majus,AP3 gene from Ilex aquifolium,MADS3 gene from Vitis vinifera,FBP3 gene from Petunia hybrida,and GGM6 gene Gnetum gnemon,respectively.These results showed that the subtractive cDNA library was constructed successfully and that the foundation was established for cloning the floral organ identity genes. 2.Isolation and phylogenetic analysis of MADS-box genes from Eustoma grandiflorumPlant MADS-box genes encode a family of highly conserved transcription factors that are involved in many different developmental processes including flower development.In order to elucidate the molecular mechanisms of floral development in a dicotyledonous species, lisianthus,total RNA prepared fron flower buds and mature flower organs,cDNA of five flower-specific MADS-box genes were isolated from this plant by the method of 3’-RACE using degenerate primers designed according to the conserved region of MADS-box protein family taken from flower bud.Sequence and phylogenetic analysis indicated that they had a high degree of identity in the predicted protein sequence with DEF gene from Antirrhinum majus,FBP3,FBP6,and FBP7 genes from Petunia,and SEP3gene from Arabidopsis thaliana,respectively.Therefore,these five genes were termed EgDEF1(DEF/GLO subfamily, B class),EgGLO1(DEF/GLO subfamily,B class),EgPLE1(AG-like subfamily,C class), EgMADS1(AGL11-like subfamily,D class),and EgSEP3-1(SEP-like subfamily,E class), respectively.Alignment of the predicted amino acid sequences from these genes,along with previously published subfamily members,demeonstrated that these genes comprise four regions of the typical MIKC-type MADS-box proteins:the MADS domain,intervening(I) domain and keratin-like(K) domain,and the C-terminal domain.Alignment of C-terminal regions of the predicted protein sequences of these genes with those of their subfamily genes revealed that each gene bears the highly conserved motifs.3.The expression of the MADS-box genes from Eustoma grandiflorumTo investigate the expression patterns of the floral organ identify MADS-box genes in flower of Eustoma grandiflorum,gene-specific RT-PCR was performed with RNA that was isolated from vegetative and floral organs.This analysis revealed that both class B-function genes EgDEF1 and EgGLO1 were strongly expressed in petals and stamens.The EgDEF1 expression was strongly detected in sepals and ovules,but the EgGLO1 expression in sepals was weakly detected with no detection in ovules.C-function gene EgPLE1 was expressed specifically in stamens,carpels,and ovules,but the expression in stamens less abundant.D-function genes EgMADS1 was expressed specifically in carpels and ovules.E-function genes EgSEP3-1 expression was strongly detected in four flower whorls.Theses experiments revealed that five MADS-box genes were expressed specifically in floral organs,while no signal was detected in vegetative organs such as root,stem,and leaf.更多还原