过量表达Fa14-3-3C促进拟南芥对低氮胁迫耐受性的研究

氮元素是植物生长发育过程必不可少的营养元素之一,对禾本科作物生长的影响更加明显。本研究采用RACE技术从高羊茅叶片中克隆获得Fa14-3-3C基因全长,并对其亚细胞定位与分子功能进行系统研究。在烟草表皮细胞中观察发现Fa14-3-3C-GFP主要定位在细胞质中与细胞膜上。将Fa14-3-3C基因在拟南芥中过量表达获得3个单拷贝转基因株系(抗性分离比为3∶1)。在低氮胁迫反应中,Fa14-3-3C过量表达株系OE-1与OE-3的根鲜重显著比野生型高,而OE-2与野生型差异不显著,通过荧光定量PCR分析发现OE-1与OE-3过量表达明显而OE-2没有过量表达,说明Fa14-3-3C对植物耐低氮胁迫调节具有剂量效应。定量观察植物根系生长发现在低氮处理早期OE-1转基因株系就显著优于野生型,主要是通过补偿根系生长的方式缓解低氮胁迫对植物的伤害。因此本研究不仅获得了耐低氮胁迫候选基因,而且验证了其在模式植物中的分子功能,为进一步通过基因工程等手段培育耐低氮胁迫种质资源奠定基础,具有重要理论研究价值与生产应用前景。

Enhanced tolerance of Arabidopsis over expressing Fa14-3-3C from tall fescue (Festuca arundinacea) to low-nitrogen stress

Nitrogen is essential for the growth and development of plants,especially gramineous crop plants.In this study,the full-length Fa14-3-3C gene was obtained by rapid amplification of cDNA ends from leaves of tall fescue (Festuca arundinacea).Subcellular localization analyses showed that Fa14-3-3C-GFP was mainly located in the cytoplasm and cell membrane when it was transiently expressed in tobacco epidermal cells.Fa14-3-3C was transferred into Arabidopsis,and three single-copy T-DNA insertion strains showing a 3 ∶ 1 hygromycin resistance segregation ratio were obtained.When wild-type and Fa14-3-3C overexpression strains were subjected to nitrogen deficiency,the root fresh weight was higher in strains OE-1 and OE-3 (but not OE-2) than in wild type.Quantitative real-time PCR analyses showed that Fa14-3-3C was highly expressed in OE-1 and OE-3,but not in OE-2,reflecting a dosage effect on the response to nitrogen deficiency.Dynamic analyses of the root growth of wild-type and Fa14-3-3C overexpression strains in nitrogen-deficient medium revealed that OE-1 showed a dramatic advantage over wild-type plants at the early stage of nitrogen deficiency.This was mainly due to compensation growth to alleviate the negative effects of low-nitrogen stress in the OE-1 strain.Therefore,we have cloned a candidate gene conferring resistance to low-nitrogen stress,and verified its molecular function in the model plant Arabidopsis.These results are fundamentally important for breeding crop plants resistant to low-nitrogen stress via genetic engineering.