【作者】 邵景侠；

【导师】 张改生； 郁飞；

【作者基本信息】 西北农林科技大学 ， 作物遗传育种， 2012， 博士

【摘要】 植物中特有的同源异型域-亮氨酸拉链(Homeodomain-leucine-zipper，HD-Zip)转录因子具有由60或61个氨基酸残基组成的高度保守的同源异型域(HD结构域)，其HD羧基末端与亮氨酸拉链(LZ)紧密相连。HD-Zip转录因子包含多个成员，在高等植物的生长、发育、形态建成，以及生物和非生物胁迫等逆境应答中起着重要的调控作用。大麦中的Vrs1基因编码一个HD-Zip I类蛋白，与大麦的六棱穗状花序有关。在二棱大麦中Vrs1参与侧部小穗的发育,其表达仅限于发育未成熟的侧部小穗原基中，Vrs1功能缺失突变导致二棱大麦未发育的侧部小穗表现出发育丰满多产的表型。序列比对结果表明水稻中的OsHOX12和OsHOX14与Vrs1的同源性较高。本研究的第一部分研究内容是从水稻数据库中搜索得到与Vrs1同源的HD-Zip I基因OsHOX12和OsHOX14。我们克隆得到这两个基因，并对其进行功能分析、表达模式分析、亚细胞定位分析及目标基因与DNA特异结合等方面进行研究，以期明确这2个基因在水稻生长发育过程中的功能。花瓣的发育主要与A类基因(AP1, AP2)、B类基因(AP3, PI)和E类基因(SEP)有关，其它一些基因(如PTL, RBE)等参与花瓣的发育。花瓣发育机理的研究具有重要的理论意义和广泛的应用前景。拟南芥花瓣发育突变体是研究花瓣发育分子机理的理想材料。本研究第二部分的研究工作是在前期利用激活标签载体pSKI015诱变的拟南芥突变体库中，筛选到一个拟南芥叶片发育突变体(abs2-1D)。通过对T-DNA插入位点的鉴定和分析，发现突变体的表型是由于At2g36080的表达量升高导致的，因此我们克隆得到ABS2基因，并对ABS2基因进行生物信息学分析、功能分析、组织表达分析及亚细胞定位等研究，以探索ABS2在拟南芥花瓣发育过程中的功能。通过上述两部分研究，获得了如下结果：（1）利用RT-PCR方法从水稻中分别得到HD-Zip转录因子家族成员OsHOX12及OsHOX14全长cDNA，序列分析和进化分析结果表明：OsHOX12和OsHOX14基因序列与大麦Vrs1基因序列同源，均属于HD-Zip家族的第一类基因，其编码蛋白都具有高度保守的HD结构域，OsHOX12和OsHOX14与Vrs1同属于一个亚类，在进化上亲缘关系较近，其中OsHOX14与Vrs1的同源性更高。（2）分别构建OsHOX12和OsHOX14的过表达载体，转化水稻与拟南芥并获得转基因植株。从DNA和RNA水平进行分子生物学检验以及遗传分析。结果表明，过表达的转基因植株均表现出生长受到抑制的表型。OsHOX12过表达植株出现分生组织上移，结实率降低等表型；转OsHOX12基因拟南芥表现为植株矮化，不能正常结实的表型；与OsHOX12过表达转基因水稻植株相比，OsHOX14过表达植株表型更为强烈，转基因植株出现包穗现象，不能结实。进一步的组织切片分析结果表明转基因植株的叶鞘和野生型有明显区别。（3）T-DNA插入失活突变体的研究表明，水稻oshox12突变体的表型与野生型相比，无明显变化；水稻oshox14突变体表现为成熟期提前等表型。（4）构建了OsHOX12和OsHOX14启动子与GUS基因的融合载体并转化水稻。GUS染色结果表明，pOsHOX12::GUS主要在成熟植株花药、内桴和颖壳中强烈表达；pOsHOX14::GUS主要在生殖器官，如花药、雌蕊等器官表达。（5）半定量RT-PCR组织特异性表达分析结果表明，OsHOX12和OsHOX14主要在开花后10天和15天的穗子中表达，在根和茎中也有表达。（6）亚细胞定位结果表明，OsHOX12和OsHOX14蛋白均定位于细胞核，符合转录因子的特性。（7）酵母单杂交和原生质体瞬时转化的结果表明，OsHOX12和OsHOX14蛋白均能与HD-Zip型转录因子的顺式作用元件DNA序列AH1(CAAT(A/T)ATTG)结合。（8）序列分析发现ABS2编码一个拟南芥B3家族转录因子，进化分析表明ABS2属于RAV亚家族，与参与拟南芥花器官发育的NGA基因亲缘关系较近。（9）构建了pBI111L::ABS2的过表达载体，转基因植物出现了与abs2-1D突变体类似的表型，并发现ABS2的过量表达导致植物花瓣缺失。（10）筛选获得了abs2T-DNA插入失活纯合突变体，突变体与野生型相比，表型上无明显变化。（11）不同组织半定量RT-PCR结果表明，ABS2在花中的表达量最高；其次是果荚和根，在其它组织中不表达。（12）亚细胞定位结果表明，ABS2蛋白定位于细胞核。（13）构建了pABS2::GUS融合载体，获得了转基因植物。更多还原

【Abstract】 The plant specific HD-Zip transcription factors include a conserved60or61amino acidsequence, known as the homeodomain (HD) motif, and a Zip motif that is immediatelyadjacent to the C-terminus of the HD motif. The HD-Zip transcription factor family membersplay vital roles in plant development, morphogenesis and plant’s responses to biotic andabiotic stresses. In barley, a recessive mutation in Vrs1changes two-rowed barley tosix-rowed barley. The Vrs1(HvHox1) gene encodes a HD-Zip transcription factor. The spatialand temporal specificity of Vrs1gene expression suggests that VRS1is a transcription factorinvolved in the development (suppression) of lateral spikelets in two-rowed barley. Sequenceanalysis showed that rice genes OsHOX12and OsHOX14were homologous of Vrs1. In thisstudy, we isolated these2novel HD-Zip I genes from rice, OsHOX12and OsHOX14, andcarried out bioinformatics analysis, functional analysis, expression pattern analysis,subcellular localization analysis and DNA-binding activity analysis to investigated thefunction of these2new transcription factor.The petal identity is determined by class A (APETALA1(AP1), APETALA2(AP2)), class B(APETALA3(AP3), PISTILLATA (PI)) and class E (SEPALLATA(SEP)) genes. Some other factors, such asPETAL LOSS (PTL) and RABBIT EARS (RBE), have been reported to be involved in the initiation andgrowth of petal primordium. Research on the mechanism of petal development can contribute to the basictheory research as well as later application, but till now the molecular mechanisms about the petaldevelopment remain poorly understood. In our previous work, we isolated an Arabidopsis B3transcriptional factor member, ABS2. Using an activation-tagging method, we isolated a RAVtranscriptional factor member, abs2-1D (abnormal shoot2-1D). In this study we found thatthe phenotype of abs2-1D was caused by NGAL1(ABS2). We cloned the ABS2gene, andcarried out bioinformatics analysi, functional analysis, tissure expression analysis and ABS2sub-cellular localization, to provide data for exploring the functional mechanism of ABS2inthe petal development.The main results are as follows:(1)The full length cDNA of OsHOX12and OsHOX14were identified from rice by RT-PCR approach, sequence and phylogenetic analysis showed that OsHOX12and OsHOX14were homologous of Vrs1, belonging to the HD-Zip subfamily I and containing typical HDdomain. OsHOX12、OsHOX14and Vrs1were in the same clade. OsHOX14is more closelywith Vrs1than OsHOX12.(2) To investigate the functions of these HD-Zip proteins, we generated OsHOX12andOsHOX14over-expression transgenic plants. Transgenic plants were identified at RNA landDNA levels by Northern and Southern blotting. The result showed that the growth of thetransgenic plants was obviously suppressed compared to the non-transgenic ones.Over-expression of OsHOX12causes apical meristem up and low fertility. Over-expression ofOsHOX14causes the panicle couldn’t come out from the sheath, and sterility. Furtherhistological section showed there were differences in the structures of sheath betweenover-expression plants and the WT.(3) oshox12T-DNA insertion mutant plants exhibited no visible phenotypic alterationscompared with the WT; oshox14mutant causes early mature.(4) The promoter regions of the OsHOX12and OsHOX14gene were fused to GUSreporter gene in pCAMBIA1391Z vector, respectively and the constructs were transformedinto rice callus and Arabidopsis by Agrobacterium-mediated transformation and floral dippingmethod. Histochemical analysis of the GUS activities indicated that pOsHOX12::GUS washighly expressed in anther, glume and palea in mature plants; pOsHOX14::GUS was mainlyexpressed in reproductive organ, such as anther and pistil.(5) Tissue expression profiles revealed by semi quantitative RT-PCR showed thatOsHOX12and OsHOX14were highly expressed in10DAF and15DAF of young panicle.OsHOX12and OsHOX14were found to be expressed in roots and stems.(6) The subcellular localization results showed that the OsHOX12and OsHOX14proteins were both localized in the nucleus.(7) Yeast one hybrid and protoplast transformation results indicated that both OsHOX12and OsHOX14could bind AH1(CAAT(A/T)ATTG) DNA sequence.(8) Phylogenetic analysis showed that ABS2belongs to the RAV subfamily of B3transcription factor, and was closely related to NGA genes，which are involved in flower organdevelopment..(9) Overexpression of ABS2showed the same phenotypes as the abs2-1D, and alsocaused petal loss phenotype,(10) There were no obvious phenotypic differences between abs2T-DNA insertionmutant and the WT. (11) Tissue expression profiles revealed by semi quantitative RT-PCR showed that ABS2was highly expressed in the flower, and it also expressed in roots and siliques, but no signialwas found in other tissues examined.(12) The sub-cellular localization results showed that the ABS2proteins was localized inthe nucleus,(13) The promoter region of the ABS2gene was fused to GUS reporter gene in pCB308vector and the construct was transformed into Arabidopsis, T1transgenic plants wereobtained.更多还原