Dorsal root ganglion progenitors differentiate to gamma-aminobutyric acid-and choline acetyltransferase-positive neurons

This study examined the isolation and differentiation of dorsal root ganglion progenitor cells for therapeutic use in neurodegenerative diseases. Rat embryonic dorsal root ganglia progenitors were isolated and purified using the differential adhesion method combined with cytosine arabinoside treatment. After culture in serum-free medium supplemented with B27, basic fibroblast growth factor and epidermal growth factor, these cells remained viable and survived for more than 18 months in vitro. Most cells differentiated to neurons that were immunoreactive for gamma-aminobutyric acid and choline acetyltransferase as detected by immunohistochemical staining. In addition, nerve growth factor and neurotrophic tyrosine kinase receptor expression were also observed in dorsal root ganglion progenitors and differentiated cells. K252a, an inhibitor that blocks nerve growth factor-induced signaling, inhibited cell survival, suggesting the possible existence of a nerve growth factor autocrine loop in these proliferating cells.

小麦生长素响应因子TaARF6转基因烟草植株分子鉴定

【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c DNA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c DNA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Bd ARF6和源于水稻的Os ARF6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。