【作者】 庄黎丽；

【导师】 罗达；

【作者基本信息】 上海交通大学 ， 遗传学， 2012， 博士

【摘要】 在植物器官发育过程中，有很多重要的调控因子参与其中，在不同的物种中，这些调控因子发挥保守的功能来控制植物器官的属性以及决定器官形状和大小。最令人感兴趣的是，在长期的进化过程中，这些因子是否获得了物种特异的功能，并且被整合到不同的分子途径中去，从而产生了自然界丰富多样的植物形态。本研究在豆科植物豌豆和百脉根中筛选了一个等位的基因座，发现这个基因座与豌豆经典遗传学中发现的一个位点LATHYROIDES (LATH)等位，lath突变以后会造成多效性的表型，影响器官中侧轴向的发育。通过比较基因组学方法克隆了LATH基因，它编码了一个含有同源异型蛋白模指的转录调控因子，其氨基酸序列与拟南芥中的WOX1高度同源；利用遗传学、细胞生物学和分子生物学等技术对LATH的功能进行研究，结果表明：LATH可以与多个发育途径的因子相互作用，参与了背部花瓣和复叶的发育。1.在豌豆和百脉根基因组中鉴别lath突变位点在豌豆快中子诱变群体中得到6个等位突变体，其表型与经典的lathyroide突变体表型类似，遗传学研究表明，它们与lath属于同一个基因座。在百脉根EMS诱变群体中得到narrow organ1(nao1)突变体。lath和nao1突变体的花瓣和叶片变窄，最明显的表型是背部花瓣变为一窄条形、失去大部分的裂片组织和细胞，且突变体雌性不育。遗传分析结果表明nao1与lath都受隐性单基因控制。2.比较基因组学克隆LATH/NAO1通过精细定位，把NAO1基因锚定在百脉根4号染色体长臂末端CM0042重叠群250K的区域。比较基因组学分析发现，该区域与蒺藜苜蓿的第4号染色体AC174277和AC137078所在区域有着非常好的微共线性，而且豌豆LATH基因也被定位到对应的共线性区域。生物信息学分析发现，共线性区域内有一个WOX1(WUSCHEL RELATEDHOMEOBOX1)同源基因，在nao1突变体中，LjWOX1发生一个核苷酸的替换导致蛋白翻译提前终止，在豌豆6个lath等位突变体中，PsWOX1基因均发生突变。3.LATH基因表达模式RT-PCR分析发现，LATH基因在豌豆地上器官均有表达，且在花苞中表达量最高，但在根中没有检测到LATH基因表达。原位杂交结果显示，LATH基因在托叶和小叶的中央维管束及两侧区域，花瓣和早期雄蕊两端及心皮的背部融合处表达；在顶端分生组织没有检测到LATH表达。4.LATH与PsCYCs在花型发育中的相互作用由于lath有强烈的背部花瓣的表型，我们在lath遗传背景下，引入了调控背部花瓣和侧部花瓣发育TCP基因（PsCYC2和PsCYC3）的突变（lst和k）构建了一系列双突变体，研究LATH与PsCYCs之间的相互作用。lath k双突变体表现为叠加的表型，lath lst双突变背部花瓣呈现lst抑制lath的表型，而侧部和腹部花瓣呈现lath突变体表型。细胞生物学分析发现：lath突变体背部花瓣缺失了典型花瓣裂片细胞区域，lst1-2突变体背部花瓣呈现侧部花瓣属性，而lath1-2lst1-2双突变体背花瓣呈现出具有背部属性的花瓣裂片细胞区。上述结果说明在豌豆花发育中，LATH与PsCYC2的遗传互作对于花瓣的背部属性有决定性作用。通过定量PCR的方法检测发现LATH与PsCYC在转录水平没有直接的调控关系。在酵母双杂交实验中也未检测到LATH与PsCYC2之间有相互作用。上述结果提示：LATH与PsCYC2的互作应该发生在转录后，但LATH与PsCYC2之间可能不发生直接的蛋白与蛋白间的相互作用。5.LATH基因参与豌豆复叶的发育LATH基因突变后，复叶中的卷须属性发生改变，与豌豆tendril-less(tl)突变体表型（小叶取代卷须）非常类似。定量PCR分析发现，lath等位突变体中Tl基因表达量均显著下调，表明：LATH基因可以通过调控TL表达，影响卷须属性和参与复叶发育的分子调控。更多还原

【Abstract】 During organ development, many key regulators have been identified in plantgenomes, which play conserved role among plant species to control the organidentities and/or determine the organ size and shape. It is intriguing thatwhether these key regulators can acquire diverse function and be integratedinto different molecular pathways among different species, giving rise to theimmense diversity of organ forms in nature. In this study, we havecharacterized and cloned LATHYROIDES (LATH), a classical locus in pea,whose mutation displays pleiotropic alteration of lateral growth of organs andpredominant effects on tendril and dorsal petal development. LATH encodesa WUSCHEL-related homeobox1(WOX1) transcription factor, which has aconserved function in determining organ lateral growth among different plant species. Furthermore, we studied the function of LATH by genetics, cellbiology and molecular biology methods and found that LATH interplay withmultiple developmental factors to regulate development of flower andcompound leaf.1. Isolation and characterization of lath and nao1mutants from pea andLotusOne narrow organ1(nao1) and6allelic mutants (lath) similar tolathyroide mutant were isolated from EMS mutagenesis in Lotus and fastneutron mutagenesis in pea respectively. Both nao1and lath had pleiotropiceffect including narrower leaves and flowers especially dorsal petal from abig petal to a narrow strip, and all the mutants except the weaker allelelath1-6were female sterile. Genetic analysis showed that nao1and lath wereboth controlled by single recessive locus.2. Molecular cloning the LATH/NAO1geneWe fine mapped NAO1to a250K region in the end of the long arm inchromosome4of Lotus. This region covered three BACs which overlappedto form CM0042contig and shared microsynteny with AC174277andAC137078in the end of chromosome4of Medicago truncatula. LATHexactly was primarily mapped to the counterpart of the region.Bioinformatics analysis found that there was a WUSCHEL RELATED HOMEOBOX1(WOX1) homologous gene in the region. Squences analysisshowed that nao1mutant carried a single base substitution in LjWOX1which putatively resulted in forming a truncated protein and PsWOX1mutated in all the six allelic mutants in pea.3. LATH expression patternRT-PCR analysis showed that LATH expressed in aerial organs andshowed the most high expression level in floral buds. But strikingly, therewas almost no or very low expression level in roots even with high cycles ofamplification. RNA in situ hybridization analysis showed that LATH mainlyexpressed in central vascular bundle in stipules and leaflets, and in the marginof leaflets and tendrils. As in floral development, LATH mainly expressed inlateral parts of five petals and early stage stamens. LATH also expressed inthe dorsal fusion region of the carpal. LATH showed no expression pattern inshoot apical meristerm (SAM).4. Genetic interaction among LATH and PsCYCsIn order to investigate the interaction between LATH and PsCYCs, wecarried out a series of genetic cross. lath k double mutant showed an additivephenotype. the lath lst double mutant showed an unexpected phenotype inthat the dorsal petal of the double mutant bore partially developed blade, andwas somewhat similar to the lst single mutant. These results suggested that LATH genetic interact with LST to regulate petal development. Narroweddorsal petal of lath losed subdomain with dorsalized epidermal cell, andlobed dorsal petal of lst altered dorsal identity with lateralized epidermal cell.However dorsal petal of lath lst somehow reaquired dorsal identiy as judgedby appearance of the dorsalized epidermal cell types.Fluorescence real-time PCR analysis showed that there was noregulation between LATH and LST in transcription level. Yeast two hybridassay also showed there was no direct interaction between LATH and LST inyeast.5. LATH regulated Tendril-less to affect tendril development in peaThe distal part of the compound leaves showed a tendril converting tonarrow leaflets phenotype in lath mutant which reminiscent of tendril-lessmutant which also showed increased leaflets but not change the complexityof the compound leaves. Fluorescence real-time PCR analysis showed thatTL was downregulated in lath mutants. This showed that LATH controlledtendril development through regulating TL gene.更多还原