燕麦AsSnRK2.7编码蛋白的分子特征及基因表达特异性研究

Molecular Characteristic Analysis and Gene Expression Specificity of AsSnRK2.7 in Oat

蔗糖非发酵相关的蛋白激酶2(SnRK2)通过磷酸化在植物胁迫信号转导途径中起关键作用。为发掘并利用燕麦中的SnRK2基因,本研究基于燕麦转录组数据的注释信息,利用RT-PCR技术从燕麦品种美达中克隆了AsSnRK2.7基因,借助生物信息学、瞬时表达及实时荧光定量RT-PCR(qRT-PCR)技术分别对该基因或其编码蛋白进行分子特征分析、亚细胞定位和表达特异性研究。结果表明,AsSnRK2.7基因包含一个1074 bp的开放阅读框,编码357个氨基酸,预测其编码蛋白含有一个STKc_SnRK2结构域和一个PKc_like superfamily结构域,属于SnRK2蛋白激酶家族成员。AsSnRK2.7蛋白一级序列中包含多个SnRK2家族特有的重要功能区。AsSnRK2.7蛋白与水稻的SnRK2蛋白激酶相似性最高,属于GroupⅠ(SnRK2b)亚家族。亚细胞定位结果显示,AsSnRK2.7蛋白主要定位在细胞核中。qRT-PCR结果显示,AsSnRK2.7基因为组成型表达,在根、茎、叶和穗中的最大表达量分别出现在苗期、分蘖期、抽穗期和灌浆期;此外,AsSnRK2.7基因的表达不被ABA激活,但可以积极应答PEG、盐和低温胁迫。以上结果说明,AsSnRK2.7基因可能作为一个调节因子,通过非依赖ABA途径来调节干旱、盐和低温所引发的信号传导。

Sucrose non-fermenting 1-related protein kinase 2(SnRK2)plays a key role in plant stress signaling pathway via phosphorylation.To explore and utilize the SnRK2 genes in oat,a SnRK2 gene from the oat variety Meida,AsSnRK2.7,was cloned based on the annotation information for transcriptome data of oat.The molecular characteristic analysis,subcellular localization,and expression specificity of the gene were investigated by bioinformatics,transient expression and real-time fluorescence quantitative RT-PCR(qRT-PCR)techniques,respectively.The results showed that the AsSnRK2.7 gene contains an open reading frame of 1074 bp encoding 357 amino acids,and its deduced protein contains an STKc_SnRK2 domain and a PKc_like superfamily domain,belonging to the SnRK2 protein kinase family.Several functional regions specific to the SnRK2 family were found to exist in primary structure of the AsSnRK2.7 protein.The AsSnRK2.7 protein showed the highest similarity with SnRK2 protein kinase in Qryza sativa,and was clustered into Group I(SnRK2b).The subcellular localization analysis revealed that the AsSnRK2.7 protein was localized primarily in the nucleus.The qRT-PCR expression analysis showed that the AsSnRK2.7 gene was expressed constitutively,and the highest relative expression in roots,stems,leaves and panicles appeared at the seedling stage,tillering stage,heading stage and filling stage,respectively.In addition,the expression of the AsSnRK2.7 gene was not activated by ABA,but its expression responded positively to PEG,NaCl and low-temperature stresses.The above results indicated that the AsSnRK2.7 gene may act as a regulator to mediate the signaling transduction triggered by dehydration,salt and low temperature through the non-dependent ABA pathway.