CAJ | 학술논문

【目的】对面包小麦(Triticum aestivum L.)中国春 NAM 转录因子 Gpc-1（TaNAM-A1、TaNAM-B1、TaNAM-D1）和 Gpc-2（TaNAM-B2、TaNAM-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cDNA 编码序列并进行序列比对分析。采用荧光定量 PCR (qRT-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 mRNA 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 TaNAM-A1、TaNAM-B2、TaNAM-D1、TaNAM-D2以及具有功能的 TaNAM-B1，且其核酸序列与野生二粒小麦（Triticum turgidum var.dicoccoides）野生型 TtNAM-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。mRNA 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。qRT-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。TaNAM-D2在各组织中表达量均最低；籽粒中 TaNAM-D1丰度最高，穗下节、颖壳及穗轴中 TaNAM-B2转录水平高于其他基因，而旗叶中 TaNAM-B2与 TaNAM-A1表达量最大。开花前，TaNAM-A1、TaNAM-B1、TaNAM-B2和 TaNAM-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 TaNAM-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 TaNAM-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，TaNAM-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， TaNAM-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 TaNAM-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。【目的】對麵包小麥(Triticum aestivum L.)中國春 NAM 轉錄因子 Gpc-1（TaNAM-A1、TaNAM-B1、TaNAM-D1）和 Gpc-2（TaNAM-B2、TaNAM-D2）灌漿期的錶達模式進行全麵繫統的分析，為深入瞭解其協同調控籽粒髮育時期組織衰老和礦物元素轉運的方式及各基因間相互作用提供參攷。【方法】從中國春中剋隆 Gpc-1和 Gpc-2的全長cDNA 編碼序列併進行序列比對分析。採用熒光定量 PCR (qRT-PCR)定量分析各基因在不同組織中的錶達特性。以多箇經評估的穩定基因作為內參，併採用 Pfaffl 法對 Gpc-1和 Gpc-2的相對錶達量進行計算。針對 Gpc-1和 Gpc-25′或3′非翻譯區（UTR）的差異覈苷痠序列設計5條特異性寡覈苷痠探針，地高辛標記後利用 mRNA 原位雜交技術分彆在花後的旂葉、穗下節及籽粒中對基因的錶達進行組織定位。【結果】利用一對特異性引物，從中國春中剋隆到 TaNAM-A1、TaNAM-B2、TaNAM-D1、TaNAM-D2以及具有功能的 TaNAM-B1，且其覈痠序列與野生二粒小麥（Triticum turgidum var.dicoccoides）野生型 TtNAM-B1完全一緻。Gpc-1和 Gpc-2的轉錄本均廣汎地分佈于倒二葉、旂葉、穗下節、穎殼、穗軸及籽粒中。但不同于 Gpc-1, Gpc-2在花後的根中併不錶達。mRNA 原位雜交結果顯示，Gpc-1和 Gpc-2具有相同的組織錶達特異性，除葉錶皮細胞、種皮和果皮外普遍在旂葉、穗下節以及籽粒的其他細胞類型中具有錶達。其轉錄本大量積纍于葉肉細胞，而在穗下節及葉片維管束中錶達量則相對較低。籽粒中 Gpc-1和Gpc-2的錶達具有不均一性，其轉錄水平在胚中較高，與礦物元素運輸相關的主要組織（維管束、色素鏈、珠心突齣及傳遞細胞）及糊粉層中次之，而胚乳中錶達量相對較低。qRT-PCR 結果顯示各 NAM 基因的錶達特性在不同組織及基因間存在差異。TaNAM-D2在各組織中錶達量均最低；籽粒中 TaNAM-D1豐度最高，穗下節、穎殼及穗軸中 TaNAM-B2轉錄水平高于其他基因，而旂葉中 TaNAM-B2與 TaNAM-A1錶達量最大。開花前，TaNAM-A1、TaNAM-B1、TaNAM-B2和 TaNAM-D2在倒二葉、旂葉、穗下節、穎殼、穗軸及籽粒中均有錶達。花後15 d，倒二葉、穗軸及籽粒中 TaNAM-A1的錶達水平先于旂葉（25DAA）齣現下降，在穗下節及穎殼中則持續增長至30DAA；籽粒中 TaNAM-B1豐度在15DAA 達到最大值，而其他組織中其最大值齣現于25DAA（旂葉、穎殼）及30DAA（倒二葉、穗下節和穗軸）；除旂葉（25DAA）及穗下節（20DAA）外，TaNAM-B2轉錄水平在其他組織中均從15DAA 起迅速降低；不同于其他基因， TaNAM-D1僅從花後開始錶達，其豐度在各分析組織中不斷增加至灌漿後期（25DAA 或30DAA）。穎殼中 TaNAM-D2錶達水平在15DAA 開始降低，早于葉片（25DAA）及其他組織。【結論】Gpc-1及 Gpc-2轉錄因子參與調控籽粒中礦物元素的轉運，但與該組織中細胞程序性死亡（programmed cell death，PCD）或衰老無顯著聯繫；灌漿期各基因間錶達特性不儘相同，其在功能上具有差異。Gpc-1和 Gpc-2或僅參與對礦物元素轉運的調控或同時平行調控組織的衰老。
【목적】대면포소맥(Triticum aestivum L.)중국춘 NAM 전록인자 Gpc-1（TaNAM-A1、TaNAM-B1、TaNAM-D1）화 Gpc-2（TaNAM-B2、TaNAM-D2）관장기적표체모식진행전면계통적분석，위심입료해기협동조공자립발육시기조직쇠로화광물원소전운적방식급각기인간상호작용제공삼고。【방법】종중국춘중극륭 Gpc-1화 Gpc-2적전장cDNA 편마서렬병진행서렬비대분석。채용형광정량 PCR (qRT-PCR)정량분석각기인재불동조직중적표체특성。이다개경평고적은정기인작위내삼，병채용 Pfaffl 법대 Gpc-1화 Gpc-2적상대표체량진행계산。침대 Gpc-1화 Gpc-25′혹3′비번역구（UTR）적차이핵감산서렬설계5조특이성과핵감산탐침，지고신표기후이용 mRNA 원위잡교기술분별재화후적기협、수하절급자립중대기인적표체진행조직정위。【결과】이용일대특이성인물，종중국춘중극륭도 TaNAM-A1、TaNAM-B2、TaNAM-D1、TaNAM-D2이급구유공능적 TaNAM-B1，차기핵산서렬여야생이립소맥（Triticum turgidum var.dicoccoides）야생형 TtNAM-B1완전일치。Gpc-1화 Gpc-2적전록본균엄범지분포우도이협、기협、수하절、영각、수축급자립중。단불동우 Gpc-1, Gpc-2재화후적근중병불표체。mRNA 원위잡교결과현시，Gpc-1화 Gpc-2구유상동적조직표체특이성，제협표피세포、충피화과피외보편재기협、수하절이급자립적기타세포류형중구유표체。기전록본대량적루우협육세포，이재수하절급협편유관속중표체량칙상대교저。자립중 Gpc-1화Gpc-2적표체구유불균일성，기전록수평재배중교고，여광물원소운수상관적주요조직（유관속、색소련、주심돌출급전체세포）급호분층중차지，이배유중표체량상대교저。qRT-PCR 결과현시각 NAM 기인적표체특성재불동조직급기인간존재차이。TaNAM-D2재각조직중표체량균최저；자립중 TaNAM-D1봉도최고，수하절、영각급수축중 TaNAM-B2전록수평고우기타기인，이기협중 TaNAM-B2여 TaNAM-A1표체량최대。개화전，TaNAM-A1、TaNAM-B1、TaNAM-B2화 TaNAM-D2재도이협、기협、수하절、영각、수축급자립중균유표체。화후15 d，도이협、수축급자립중 TaNAM-A1적표체수평선우기협（25DAA）출현하강，재수하절급영각중칙지속증장지30DAA；자립중 TaNAM-B1봉도재15DAA 체도최대치，이기타조직중기최대치출현우25DAA（기협、영각）급30DAA（도이협、수하절화수축）；제기협（25DAA）급수하절（20DAA）외，TaNAM-B2전록수평재기타조직중균종15DAA 기신속강저；불동우기타기인， TaNAM-D1부종화후개시표체，기봉도재각분석조직중불단증가지관장후기（25DAA 혹30DAA）。영각중 TaNAM-D2표체수평재15DAA 개시강저，조우협편（25DAA）급기타조직。【결론】Gpc-1급 Gpc-2전록인자삼여조공자립중광물원소적전운，단여해조직중세포정서성사망（programmed cell death，PCD）혹쇠로무현저련계；관장기각기인간표체특성불진상동，기재공능상구유차이。Gpc-1화 Gpc-2혹부삼여대광물원소전운적조공혹동시평행조공조직적쇠로。
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.