小麦生长素响应因子 TaARF6转基因烟草植株分子鉴定

小麦生长素响应因子 TaARF6转基因烟草植株分子鉴定智一鸣;陈芳;刘晓曼;肖凯【摘要】【目的】生长素响应因子(ARF)在介导生长素信号传递和调控下游生长素响应基因的表达中发挥着重要功能。

本文旨在以在富集丰磷特异表达基因的小麦根系 cDNA 差减文库中鉴定的1个 ARF 类别的家族成员 TaARF6为基础,对该基因cDNA 序列、分子特征、不同供磷水平下该基因在根、叶中表达模式及遗传转化TaARF6对丰磷和缺磷条件下植株形态的影响进行较全面研究,阐明该小麦生长素响应因子基因介导不同供磷水平下对植株生长特性的影响。

【方法】采用生物信息学工具预测 TaARF6编码蛋白特征；采用溶液培养法培养丰、缺磷处理小麦幼苗,采用半定量 RT-PCR 技术鉴定 TaARF6在丰、缺磷处理下的表达特征。

采用 DNA 重组技术构建将 TaARF6编码阅读框融合至表达载体中的表达质粒,利用农杆菌介导的遗传转化法建立超表达 TaARF6转基因烟草植株。

采用琼脂培养和溶液培养法,培养丰、缺磷不同供磷水平下野生型植株和转基因烟草植株,进而利用常规分析方法鉴定不同磷水平下植株长势、根系和茎叶生物量和植株根叶形态及性状。

【结果】1)TaARF6编码生长素响应因子(ARF)型转录因子,编码蛋白中含有 ARF 家族成员具有的保守结构域。

该基因在氨基酸水平上与源于短柄草 BdARF6和源于水稻的 OsARF6具有高度同源特征。

表达分析表明,TaARF6在根、叶中均呈典型低磷下表达下调、复磷后表达再度回升模式,表明该基因表达受到外界供磷水平的调节。

2)遗传转化结果表明,在正常生长和低磷逆境下,与野生型植株相比,转基因烟草株系幼苗和植株形态明显增大。

3)丰、缺磷不同供磷水平下,与未转化的野生型(WT)对照植株相比,转基因系(Line 3和 Line 5)植株幼苗和植株根系、茎叶和单株鲜、干重均较野生型显著增加。

此外,与 WT 相比,转基因系植株根系数量增多、主侧根长度、根体积、叶面积和根冠比增加。

【结论】TaARF6编码典型的生长素响应因子,其编码蛋白具有生长素响应因子特有结构域。

TaARF6对环境中的低磷胁迫逆境能产生明显应答。

上调表达 TaARF6基因,具有增加植株根、叶鲜、干重和改善根叶及植株形态的生物学功能。

本研究表明,通过对植株体内生长素响应基因的转录调控,TaARF6在介导植株不同供磷水平下的根叶形态建成和干物质累积过程中发挥着重要作用。

%[Objectives] Auxin response factors (ARFs) play a critical role in mediating transduction of auxin signaling and regulating expression of downstream auxin-responsive genes. In this study, an ARF type transcription factor gene referred to TaARF6 identified in a root suppression subtractive cDNA library that enriches the differentially expressed genes under Pi sufficiency as the basis was used to investigate molecular characterization of TaARF6 and its expression patterns under various Pi-supply conditions, as well as its functions in regulating plant phenotypes. The objective of this study was aimed to elucidate biological roles of TaARF6 in mediating plant growth features under the conditions of Pi sufficiency and Pi deficiency. [ Methods] The protein characterization of TaARF6 was predicted by the bioinformatics’ tools. The seedlings of wheat (cv. Shixin 828) were cultured under the sufficient-and deficient-Pi conditions by a hydroponic approach and used to investigate the expression patterns of TaARF6 based on semi-quantitative RT-PCR. A DNA recombinant technique was adopted to construct the expression cassette integrating the TaARF6 open reading frame. The transgenic tobacco plants overexpressing TaARF6 were generated based on a genetic transformation approach mediated by Agobacterium-tumefaciens using the leaf discs as explants. Based on the culture methods of agar medium and hydroponicsolution, the seedlings and plants of the transgenic Line 3 and Line 5 as well as wild type (WT) were cultured to determine the growth features and to assay the fresh and dry weights of roots and aboveground tissues under the conditions of Pi sufficiency and Pi deficiency. [Results] 1) TaARF6 encodes a transcription factor categoried into auxin response factor family and shares the conserved domains of ARF proteins in plants, such as the DNA binding domain and the auxin response domain. It shares a high identity to BdARF6 and OsARF6, two ARF family members in B. distachyon and rice, respectively, at the amino acid level. The expression analysis reveals that TaARF6 is significantly down-regulated by Pi deprivation in roots and leaves, and its expression in the above tissues is restored by a recovery treatment initiated by retransferring the Pi-deprived plants to normal-Pi condition. 2) Compared with the wild type, the tobacco seedlings and plants overexpressing TaARF6 are improved under both Pi sufficiency and Pi deficiency conditions. 3) Compared with the wild type, the fresh and dry weights in roots, stems and leaves, and in the whole plant of the tobacco seedlings and plants with overexpression of TaARF6 are increased under the conditions of both normal growth and Pi deprivation, and the lateral root numbers, lengths of the primary and lateral roots, root volume, leaf areas and the ratios of root weight to aboveground tissue weight are also increased. [Conclusions]Our results in this study confirm that TaARF6 encodes a typical auxin response factor, and its encoded protein contains distinct domains generally situated in ARFs. TaARF6 is significantly responsive to external Pi levels.Overexpression of TaARF6 can improve plant growth features, fresh and dry weights under the conditions of normal growth and Pi deprivation. Together, our investigation in this study suggests that TaARF6 plays critical roles in mediating plant growth and dry mass production under various Pi-supply conditions, which is possibly finished through its transcriptional regulating expression of the downstream auxin-responsive genes.【期刊名称】《植物营养与肥料学报》【年(卷),期】2016(022)001【总页数】9页(P85-93)【关键词】小麦(Triticum aestivum L.);生长素响应因子;分子特征;遗传转化;根叶生长【作者】智一鸣;陈芳;刘晓曼;肖凯【作者单位】河北农业大学农学院，河北保定 071001;河北农业大学农学院，河北保定 071001;河北农业大学农学院，河北保定 071001;河北农业大学农学院，河北保定 071001【正文语种】中文【中图分类】S512.1.01;Q954.78genetic transformation; root and leaf growth生长素是植物生长发育的重要激素类物质，生长素信号与植株顶端优势、根系侧根发生、维管组织分化等密切相关[1-2]。