CAJ | 학술논문

光周期是植物从营养生长转为生殖生长的重要影响因子，CO(constans)基因在模式植物拟南芥(Arabidopsis thaliana)的光周期途径中起重要作用。本研究利用同源克隆技术从普通小麦(Triticum aestivum L.)宁春4号中克隆了CO同源基因TaCO9-1A(GenBank登录号：KM236233)的gDNA(genomic DNA)及cDNA(complementary DNA)。TaCO9-1A的全长编码区(coding sequences, CDS)为876 bp，编码291个氨基酸，含有CO-like蛋白家族典型的CCT结构域，但不含B-box结构域；系统进化分析表明， TaCO9-1A蛋白与水稻(Oryza sativa L.)Ghd7及大麦(Hordeum vulgare L.)HvCO9位于同一分支；蛋白质空间结构分析表明，其CCT结构域的NF-YA2区域较为保守；TaCO9-1A原核表达蛋白分子量为31 kD，与预测结果一致；实时荧光定量PCR结果显示，TaCO9-1A在普通小麦抽穗期的根、茎、叶及幼穗均有表达，但根部表达量较低，相对表达量依次为叶＞茎＞幼穗＞根。比较该基因在冬春性不同品种中的核酸序列发现，冬性品种第二外显子存在6个碱基的缺失，针对该差异开发了分子标记，在25份冬春性不同的小麦品种中扩增出511和517 bp两种带型，分别与这些品种的冬春性及在杨凌地区两年的抽穗及开花时间早晚显著相关。本研究结果表明，TaCO9-1A在小麦春化作用和光周期途径中扮演着重要角色，为研究小麦光周期途径和春化作用途径提供了基础资料，有助于揭示TaCO9-1A调控小麦冬春性及成熟期的分子机理。
광주기시식물종영양생장전위생식생장적중요영향인자，CO(constans)기인재모식식물의남개(Arabidopsis thaliana)적광주기도경중기중요작용。본연구이용동원극륭기술종보통소맥(Triticum aestivum L.)저춘4호중극륭료CO동원기인TaCO9-1A(GenBank등록호：KM236233)적gDNA(genomic DNA)급cDNA(complementary DNA)。TaCO9-1A적전장편마구(coding sequences, CDS)위876 bp，편마291개안기산，함유CO-like단백가족전형적CCT결구역，단불함B-box결구역；계통진화분석표명， TaCO9-1A단백여수도(Oryza sativa L.)Ghd7급대맥(Hordeum vulgare L.)HvCO9위우동일분지；단백질공간결구분석표명，기CCT결구역적NF-YA2구역교위보수；TaCO9-1A원핵표체단백분자량위31 kD，여예측결과일치；실시형광정량PCR결과현시，TaCO9-1A재보통소맥추수기적근、경、협급유수균유표체，단근부표체량교저，상대표체량의차위협＞경＞유수＞근。비교해기인재동춘성불동품충중적핵산서렬발현，동성품충제이외현자존재6개감기적결실，침대해차이개발료분자표기，재25빈동춘성불동적소맥품충중확증출511화517 bp량충대형，분별여저사품충적동춘성급재양릉지구량년적추수급개화시간조만현저상관。본연구결과표명，TaCO9-1A재소맥춘화작용화광주기도경중분연착중요각색，위연구소맥광주기도경화춘화작용도경제공료기출자료，유조우게시TaCO9-1A조공소맥동춘성급성숙기적분자궤리。
Photoperiod plays an important role in triggering the change from vegetative growth to reproductive growth. CO (constans) plays a significant role in the photoperiod pathway in the model plant Arabidopsis. To learn more about the CO-like gene in wheat, the homologous cloning method was used to isolate the related gene. Here a novel gene TaCO9-1A (GenBank accession:KM236233) that encoding a CO-like protein was cloned from hexaploid wheat (Triticum aestivum L.) Ningchun 4. The results showed that the coding sequences (CDS) of TaCO9-1A was 876 bp in length, encoding 291 amino acids including a classic CCT domain, but not the B-box domain. A phylogenetic analysis showed that TaCO9-1A was classified into the same branch with Ghd7 in rice (Oryza sativa L.) and HvCO9 in barely (Hordeum vulgare L.), which were negative regulators of flowering. TaCO9-1A protein with a predicted molecular weight 31 kD was succesfully expressed by the plasmid pEASYE1-TaCO9-1A in Escherichia coli BL21 (DE3). The expressions of TaCO9-1A were detected in root, stem, leaf and young spike in common wheat at heading stage by Real-time quantitative PCR (qRT-PCR), and the expression level was highest in leaf and lowest in root. Comparing the sequence of TaCO9-1A in winter and spring varieties, it was found that 6 bp deletion in the second exon existed in winter varieties. Thus, a codominant marker was developed based on the 6 bp deletion of TaCO9-1A in winter varieties. In 25 wheat varieties with different winter and spring habits, two kinds of bands (511 and 517 bp) were amplified with this marker. The codominant marker was highly correlated with not only spring and winter habit but also the heading, flowering time of wheat cultivated at Yangling in two years. Our results revealed that TaCO9-1A played an important role in wheat vernalization and photoperiod pathway. These results provide basic data for the further research of TaCO9-1A regulating development in wheat, and will help to reveal the molecular mechanism of spring-winter habit and maturity traits in wheat by TaCO9-1A regulation.