发菜PspA基因克隆及其转基因植物的抗旱性分析

Isolation of the PspA Gene from Nostoc flagelliforme and Analysis of Its Drought Resistance in Transgenic Plants

为探讨发菜噬菌体休克蛋白A(PspA)的分子信息和基因功能,本研究通过设计特异引物克隆发菜PspA基因,采用qRT-PCR技术,分析发菜PspA基因在干旱胁迫下的表达模式;构建PspA真核表达载体pCAM35s-GFP-PspA,对PspA进行亚细胞定位和PspA基因拟南芥遗传转化,并对阳性转化拟南芥分别进行Southern和Western杂交验证;对转基因植株进行抗旱实验,结果表明,PspA基因全长为777 bp,干旱胁迫下发菜PspA基因表达量显著增加;PspA定位于细胞膜上,通过花絮浸染法获得稳定遗传的转PspA基因拟南芥。Southern杂交表明,PspA基因已成功导入拟南芥基因组中并以低拷贝形式存在,Western blot进一步证实PspA蛋白在转基因拟南芥中成功表达。在干旱胁迫下,转PspA基因拟南芥生长状态明显好于野生型植株。研究结果为深入探讨发菜PspA基因功能及其在响应干旱胁迫过程中的应答机制奠定了基础。

In order to study the functional basis of Phage shock protein A(PspA),the full-length coding sequence(CDS)of the PspA gene was isolated based on sequence homolog and its expression pattern of PspA from Nostoc flagelliforme Born.&Flah.under drought stress was studied by RT-qPCR.We further generated a plasmid pCAM35 s-GFP-PspA which were subjected for a subcellular localization analysis and a transformation in Arabidopsis thaliana(L.)Heynh.PspA contained a 777 bp full-length coding sequence,and this gene was significantly up-regulated in N.flagelliforme Born.&Flah.upon drought stress treatment.A transient expression of PspA in N.benthamiana Domin suggested a sublocalization on the plasma membrane.Moreover,by transforming this gene in A.thaliana(L.)Heynh.,the transgenic plants showed low copies of the PspA gene using Southern hybridization and expressed the recombination protein by Western blot analysis.The transgenic plants expressing the PspA gene,if compared to the wild type plants,were found to be highly resistant against drought stress.Collectively,the results provided a foundation for further exploring the biological function of the N.flagelliforme Born.&Flah.PspA gene that interplays with drought stress.