小麦光周期基因TaCO9-1A的克隆、功能标记开发及其与冬春性关系的研究  

Molecular Cloning and Functional Maker Design of Ta CO9-1A Gene Associated with Spring-winter Habit in Wheat (Triticum aestivum L.)

作　　者：冉从福[1] 邵慧[1] 余静[1] 寇程[1] 李扬[1] 李立群 李学军[1] 

机构地区：[1]西北农林科技大学农学院/旱区作物逆境生物学国家重点实验室,杨凌712100

出　　处：《农业生物技术学报》2014年第12期1471-1481,共11页Journal of Agricultural Biotechnology

基　　金：农业部重大专项(No.2013ZX08002-003);陕西省科技统筹创新工程计划项目(No.2014KTZB02-01-01);国家高技术研究发展计划(863)项目(No.2011AA100501)

摘　　要：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Ta CO9-1A(Gen Bank登录号:KM236233)的g DNA(genomic DNA)及c DNA(complementary DNA)。Ta CO9-1A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,Ta CO9-1A蛋白与水稻(Oryza sativa L.)Ghd7及大麦(Hordeum vulgare L.)Hv CO9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;Ta CO9-1A原核表达蛋白分子量为31 k D,与预测结果一致;实时荧光定量PCR结果显示,Ta CO9-1A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,Ta CO9-1A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Ta CO9-1A调控小麦冬春性及成熟期的分子机理。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 intothe 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 mechan

关 键 词：小麦光周期基因 TaCO9—1A 同源克隆 分子标记 冬春性 

分 类 号：S512.1[农业科学—作物学]