大豆GmRSM1通过调节PIN基因表达促进顶端弯钩消失

Soybean GmRSM1 promotes apical hook disappearance by regulating PIN gene expression

顶端弯钩是双子叶植物黄化幼苗短暂出现的独有结构,尽管当前拟南芥顶端弯钩的模型网络已经相对透彻,但在大豆中的研究却非常少。本试验构建pBAR-GmRSM1过表达载体,通过遗传转化和阳性植株筛选,得到3个拟南芥过表达纯合株系(OE-GmRSM1#64、#69和#70)和3个大豆纯合株系(OE-GmRSM1#103、#78和#95),转化体的表达量均显著高于野生型。暗培养拟南芥和大豆,过表达转化体的顶端弯钩消失或消失速度快于野生型。在扫描电镜下观察拟南芥野生型和转化体植株顶端弯钩部位细胞长度发现,野生型在弯钩维持阶段弯钩内侧细胞长度小于外侧,而转化体植株两侧细胞长度相同。分离大豆顶端弯钩内外两侧,并通过检测PIN基因在大豆野生型与3个过表达转化体顶端弯钩的表达情况,发现PIN1e、PIN3d和PIN6a三个基因在子叶出土时的顶端弯钩部位表达量显著高于顶端弯钩展开后的表达量,而转化体中弯钩处这3个基因在弯钩维持阶段的表达量显著高于野生型,但在展开后的表达量并无显著性差异。经过烟草瞬时表达,确定GmRSM1蛋白定位在细胞核和细胞膜。因此,顶端弯钩出现的原因是弯钩内外两侧细胞差异伸长,因而导致下胚轴弯曲。GmRSM1可以通过正向调控生长素运输载体PIN1e、PIN3d和PIN6a缩短弯钩维持和展开时间,验证了该基因对生长素转运的调控作用。本试验通过验证GmRSM1基因在顶端弯钩消失表型中的作用,进一步完善了顶端弯钩的基因通路,为后续研究奠定基础。

The apical hook is a transient structure found in etiolated dicot seedlings,and while the current model for the apical hook in Arabidopsis thaliana is relatively well-established,there is limited research on soybean.In this study,the pBAR-GmRSM1 overexpression vector was constructed,and three homozygous lines each of Arabidopsis thaliana(OE-GmRSM1#64,#69,and#70)and soybean(OE-GmRSM1#103,#78,and#95)were generated through genetic transformation and positive plant selection.The expression levels of the transgenic lines were significantly higher than the wild type.Both Arabidopsis thaliana and soybean cultures were grown in darkness,and the apical hooks in the overexpressed transformants disappeared or exhibited faster disappearance than the wild type.Scanning electron microscopy observations of cell length in the apical hook of Arabidopsis thaliana wild-type and transformants revealed that the inner hook cells were shorter than the outer hook cells during the hook maintenance stage,while the cell lengths were same on both sides in the transformant plants.The inner and outer sides of the soybean apical hook were separated,and the expression of the PIN gene in the wild type and three overexpressed transformants of soybean was analyzed.The results demonstrated that the expression levels of the PIN1e,PIN3d,and PIN6a genes were signifi-cantly higher in the apical hook after cotyledon unfolding,and during the hook maintenance stage,these three genes exhibited significantly higher expression levels in the transformants than the wild type,but there was no significant difference in expression levels after unfolding.Transient expression in tobacco confirmed than the GmRSM1 protein were localized in the nucleus and cell membrane.Thus,the apical hook formation is attributed to differential cell elongation between the inner and outer sides of the hook,leading to hypocotyl curvature.GmRSM1 regulates the expression of auxin transporters PIN1e,PIN3d,and PIN6a,thereby shortening the duration of hook maintenance and unfolding.These genes are positively regulated by GmRSM1 during the hook stage,and the study confirmed the regulatory effect of this gene on auxin transport.Overall,this study verified the role of GmRSM1 in the disappearance of the apical hook phenotype and further elucidated the gene pathway involved in apical curvature,laying a foundation for future research.