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

【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转录水平高于其他基因,而旗叶中Ta NAM-B2与Ta NAM-A1表达量最大。开花前,Ta NAM-A1、Ta NAM-B1、Ta NAM-B2和Ta NAM-D2在倒二叶、旗叶、穗下节、颖壳、穗轴及籽粒中均有表达。花后15 d,倒二叶、穗轴及籽粒中Ta NAM-A1的表达水平先于旗叶(25DAA)出现下降,在穗下节及颖壳中则持续增长至30DAA;籽粒中Ta NAM-B1丰度在15DAA达到最大值,而其他组织中其最大值出现于25DAA(旗叶、颖壳)及30DAA(倒二叶、穗下节和穗轴);除旗叶(25DAA)及穗下节(20DAA)外,Ta NAM-B2转录水平在其他组织中均从15DAA起迅速降低;不同于其他基因,Ta NAM-D1仅从花后开始表达,其丰度在各分析组织中不断增加至灌浆后期(25DAA或30DAA)。颖壳中Ta NAM-D2表达水平在15DAA开始降低,早于叶片(25DAA)及其他组织。【结论】Gpc-1及Gpc-2转录因子参与调控籽粒中矿物元素的转运,但与该组织中细胞程序性死亡(programmed cell death,PCD)或衰老无显著联系;灌浆期各基因间表达特性不尽相同,其在功能上具有差异。Gpc-1和Gpc-2或仅参与对矿物元素转运的调控或同时平行调控组织的衰老。

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 upon the organs and the genes. According to the Ct value, TaNAM-D2 was expressed less than other genes in all tested tissues. And the most abundant gene in wheat kernel was TaNAM-D1, whereas that in peduncle, glume and the rachis was TaNAM-B2. Besides, the expression levels of TaNAM-B2 and TaNAM-A1 were the highest in flag leaf among five NAM genes. TaNAM-A1, TaNAM-B1, TaNAM-B2 and TaNAM-D2 were expressed in all studied tissues before anthesis, whereas TaNAM-D1 was expressed only since the first-flowering date. TaNAM-A1 exhibited the distinct expression patterns among the tissues. Its transcript abundance began to decline in penultimate leaf, rachis and the kernel at 15 DAA, which was preceded that in flag leaf （25 DAA） and other tissues. The transcription levels of TaNAM-B1 and TaNAM-D2 kept increasing till the late filling stage （25 DAA or 30 DAA） in most organisms except for advanced declines of TaNAM-B1 and TaNAM-D2 in the grain and the glume, respectively. The expression level of TaNAM-B2 decreased from 15DAA in most tested tissues other than the flag leaf （25 DAA） and the peduncle （20 DAA）. Unlike other genes, a consistent growth until 25 DAA and 30DAA in transcript abundance of TaNAM-D1 was observed. [Conclusion] In conclusion, Gpc-1 and Gpc-2 were closely associated with mineral translocation in the grain; however, no obvious relationship between five NAM genes and the PCD in wheat grain was observed. And the discrepant temporal expression dynamics suggested that their functions were not identical. In addition, the potential effect of these genes on senescence in vegetative tissues was still elusive. Combined with previous findings, it was proposed that Gpc-1 and Gpc-2 may directly regulate nutrient remobilization only or in parallel with the senescence during grain filling.