小麦Tae-miR167克隆及抗旱功能分析

Clone and drought resistance function analysis of wheat Tae-miR167

关键词:  小麦, miRNA, 干旱, 过表达, 功能

Key words:  wheat, miRNA, drought, over-expression, function

摘要:

miR167家族是植物界中一个保守的miRNA家族,在植物生长发育及逆境胁迫响应中发挥重要调控作用。为了明确小麦 Tae-miR167 在逆境胁迫中的功能,该研究对小麦Tae-miR167家族序列进行了鉴定及序列分析,通过qRT-PCR方法分析Tae-miR167成熟体在小麦不同器官中的差异性表达和逆境胁迫表达情况; 克隆Tae-miR167c前体序列,构建过表达载体转化拟南芥,研究过表达Tae-miR167c株系对干旱胁迫响应的功能。结果表明:(1)Tae-miR167家族共有18个序列,成熟序列有3种,二级结构都有典型茎环结构。(2)Tae-miR167的3种成熟体在小麦多数器官中都有表达,其相对表达量在根、叶和种子中较高; 成熟体Tae-miR167b和Tae-miR167c可分别响应低温和PEG胁迫处理,表达上调。(3)过表达Tae-miR167c前体的拟南芥株系在渗透胁迫下发芽率和根长显著增大; 转基因幼苗耐旱性增强,含水量、可溶性糖含量、叶绿素含量显著升高。(4)靶基因预测显示,Tae-miR167c可以与F-box蛋白结合,参与调控逆境胁迫响应。综上表明,小麦Tae-miR167c在干旱胁迫下显著上调,过表达Tae-miR167c的转基因株系增强了干旱胁迫耐受性。该研究结果丰富了Tae-miR167的功能研究,可为小麦种质创新提供新的基因资源。

Abstract:

The miR167 family is a conserved microRNA(miRNA)family in plants, which plays a vital role in regulating plant growth, development, and abiotic stress response. To clarify the function of wheat miR167 under abiotic stress, the Tae-miR167 family sequences in wheat were identified and analyzed. The differential expression of Tae-miR167 mature in various wheat organs and in response to abiotic stresses were analyzed using quantitative reverse transcription polymerase chain reaction(qRT-PCR). The precursor sequence of Tae-miR167c was cloned, and its function in drought stress response was investigated through over-expression in Arabidopsis thaliana. The results were as follows:(1)The Tae-miR167 family comprised 18 members, which gave rise to three mature miRNA sequences. Tae-miR167 exhibited a characteristic hairpin structure.(2)The three matures of Tae-miR167 were expressed in most wheat organs, with relatively higher expression levels observed in roots, leaves, and seeds. The expressiones of Tae-miR167b and Tae-miR167c matures were up-regulated in response to low temperature and PEG-induced drought stress treatments, respectively.(3)The A. thaliana lines of over-expression Tae-miR167c precursor significantly improred germination rates and root lengths under osmotic stress. Additionally, the drought tolerance of these transgenic seedlings was improved, and significant increases were observed in water content, soluble sugar content, and chlorophyll content.(4)Target gene prediction indicated that Tae-miR167c could bind to an F-box protein, thereby participating in the regulation of abiotic stress response. In conclusion, miR167c was significantly up-regulated under drought stress, and over-expression Tae-miR167c transgenic lines enhanced tolerance to drought stress. This study enhances the understanding of the function of wheat miR167 and provides novel genetic resources for wheat germplasm innovation.