小麦中国春NAM转录因子Gpc-1和Gpc-2灌浆期时空表达模式分析  

Spatiotemporal Expression Pattern Analysis of NAM Transcription Factors Gpc-1 and Gpc-2 in Bread Wheat Cultivar Chinese Spring During Grain Filling

作　　者：吴丹[1] 董剑[1,2] 要燕杰 赵万春[1] 高翔[1,2] 

机构地区：[1]西北农林科技大学农学院,陕西杨凌712100 [2]陕西省小麦工程技术研究中心/陕西省小麦新品种培育研究中心,陕西杨凌712100

出　　处：《中国农业科学》2015年第7期1262-1276,共15页Scientia Agricultura Sinica

基　　金：国家"十二五"科技支撑计划农村领域课题(2011AA100501);国家现代农业产业技术体系建设专项(CARS-3-2-47)

摘　　要：【目的】对面包小麦(Triticum aestivum L.)中国春NAM转录因子Gpc-1(Ta NAM-A1、Ta NAM-B1、Ta NAM-D1)和Gpc-2(Ta NAM-B2、Ta NAM-D2)灌浆期的表达模式进行全面系统的分析,为深入了解其协同调控籽粒发育时期组织衰老和矿物元素转运的方式及各基因间相互作用提供参考。【方法】从中国春中克隆Gpc-1和Gpc-2的全长c DNA编码序列并进行序列比对分析。采用荧光定量PCR(q RT-PCR)定量分析各基因在不同组织中的表达特性。以多个经评估的稳定基因作为内参,并采用Pfaffl法对Gpc-1和Gpc-2的相对表达量进行计算。针对Gpc-1和Gpc-25′或3′非翻译区(UTR)的差异核苷酸序列设计5条特异性寡核苷酸探针,地高辛标记后利用m RNA原位杂交技术分别在花后的旗叶、穗下节及籽粒中对基因的表达进行组织定位。【结果】利用一对特异性引物,从中国春中克隆到Ta NAM-A1、Ta NAM-B2、Ta NAM-D1、Ta NAM-D2以及具有功能的Ta NAM-B1,且其核酸序列与野生二粒小麦(Triticum turgidum var.dicoccoides)野生型Tt NAM-B1完全一致。Gpc-1和Gpc-2的转录本均广泛地分布于倒二叶、旗叶、穗下节、颖壳、穗轴及籽粒中。但不同于Gpc-1,Gpc-2在花后的根中并不表达。m RNA原位杂交结果显示,Gpc-1和Gpc-2具有相同的组织表达特异性,除叶表皮细胞、种皮和果皮外普遍在旗叶、穗下节以及籽粒的其他细胞类型中具有表达。其转录本大量积累于叶肉细胞,而在穗下节及叶片维管束中表达量则相对较低。籽粒中Gpc-1和Gpc-2的表达具有不均一性,其转录水平在胚中较高,与矿物元素运输相关的主要组织(维管束、色素链、珠心突出及传递细胞)及糊粉层中次之,而胚乳中表达量相对较低。q RT-PCR结果显示各NAM基因的表达特性在不同组织及基因间存在差异。Ta NAM-D2在各组织中表达量均最低;籽粒中Ta NAM-D1丰度最高,穗下节、颖壳及穗轴中Ta NAM-B2转录水平高于其他�Objective]The objective of this experiment is to study the roles of no apical meristem （NAM） transcription factors Gpc-1 and Gpc-2 in early senescence and nutrient remobilization to the grain of bread wheat. [Method] Their spatiotemporal expression patterns were investigated during the grain-filling stage in wheat cultivar Chinese Spring. Their temporal expression dynamics were studied in penultimate leaf, flag leaf, peduncle, glume, rachis and the kernel using quantitative real time polymerase chain reaction （qRT-PCR）. And the relative expression level was quantified using Pfaffl method with normalization against multiple verified reference genes. Applying mRNA in situ hybridization, the spatial expression pattern was explored in post-anthesis flag leaf, peduncle and the kernel only with digoxin-labeled oligonucleotide probes which were specifically targeting 5′ or 3′ untranslated regions （UTRs） of Gpc-1 and Gpc-2. [Result] Contrary to a previous report, the functional TaNAM-B1 rather than its dysfunctional paralog was found in Chinese Spring, and its nucleotide sequence was identical with the wild-type TtNAM-B1 in T. turgidum var.dicoccoides. All the results showed that Gpc-1 and Gpc-2 were all widely expressed in studied tissues with the exception of the root in which only the transcript of Gpc-1 was detected. The outcomes of mRNA in situ hybridization indicated that all five genes shared cell-type specificities. To be specific, no transcripts were distributed in leaf epidermal cells, pericarp and the seed coat;however, they mainly aggregated in leaf mesophyll cells, aleurone layer, embryo, and the tissues responsible for the mineral element transport （vascular bundle, pigment strand, nucellar projection and the transfer cell） in grain, in which the highest expression level was observed in embryo. In addition, lower expression level was detected in the peduncle and leaf vascular bundle as well. The results of qRT-PCR showed that the temporal expression dynamics of Gpc-1 and Gpc-2 differed u

关 键 词：小麦 灌浆期 衰老 物质转运 表达模式 调控 

分 类 号：S512.1[农业科学—作物学]