海马齿Spmet基因表达产物的亚细胞定位及其组织表达特异性分析

Sub-cellular Localization and Expression Analysis of Spmet in Sesuvium portulacastrum L.

为研究从海马齿(Sesuvium portulacastrumandL.)中克隆的Spmet基因在植株体内亚细胞水平上的分布情况及组织表达的特异性,将Spmet基因的编码区定向克隆至植物表达载体pCAMBIA-1304上,使其与mgfp(绿色荧光蛋白)基因融合,构建Spmet基因植物瞬时表达载体,使用基因枪法将该表达载体导入洋葱表皮细胞,通过荧光激发,在荧光显微镜下观察洋葱表皮细胞中的绿色荧光部位,确定其在细胞中的表达部位。同时利用半定量PCR的方法对其在根、茎、叶中的表达特性进行分析。结果表明,该基因在海马齿植物的根、茎、叶中均有表达,表达量为叶>茎>根,融合蛋白主要分布在洋葱表皮细胞的细胞核上,细胞膜上也有微量表达。说明海马齿2型金属硫蛋白基因在海马齿植物中属于组成型表达,同时是一个核表达的基因,该结果为进一步研究Spmet基因在抗重金属方面发挥的作用打下了基础。

In order to study the Spmet distribution on subcellular level and the specificity of tissue expression, the coding region of Spmet gene was directional cloned on plant expression vector pCAMBIA-1304, and fused with fluorescent protein gene mgfp, to construct plant instantaneous expression vector. Then this vector was transferred to onion epidermal cell by gene gun bombardment, after fluorescence excitation, green fluorescence parts in onion epidermal were observed under fluorescence microscope, and then the expression sites of Spmet could be confirmed. Semi-quantitative PCR was used to analyze the expression characteristics of Spmet gene in roots, stalks and leaves, and the results showed that, the genes expression in roots, stalks and leaves, and the expression level was leaf stalk root. The fusion protein was distributed mainly on the nucleus of onion epidermal cell, and merely on cell membranes. It suggested that the gene of metallothionein 2 in Sesuvium portulacastrumand expressed constitutively, and mainly in nucleus. This result laid a foundation for further study of Spmet gene in heavy metal resistance.