丹参Remorin基因SmREM1.2的克隆及生物信息学和表达分析

Cloning and Bioinformatics and Expression Analyses of Remorin Gene SmREM1.2 from Salvia miltiorrhiza Bunge

Remorin蛋白是一类植物特异的寡聚丝状蛋白,定位于细胞膜,介导脂筏微区形成,在植物逆境胁迫应答及植物免疫调节等过程中发挥重要作用。丹参是我国大宗类中药材,广泛用于心脑血管疾病等的治疗。但目前丹参Remorin蛋白的相关研究报道还不多。本研究基于丹参转录组及基因组数据库,克隆得到丹参Remorin基因SmREM1.2,通过生物信息学方法对其氨基酸组成、保守序列、系统进化进行分析,并对其亚细胞定位及在盐胁迫下的表达情况进行分析。结果表明,该基因CDS全长585 bp,编码194个氨基酸残基。SmREM1.2蛋白为弱酸性不稳定的亲水性蛋白,无信号肽,无跨膜区,含有Remorin_N和Remorin_C保守结构域,二级结构以α螺旋和无规则卷曲为主;氨基酸序列分析结果显示其C端具有高度保守的coiled-coil模体,属于典型的REM蛋白;系统进化树分析结果表明SmREM1.2与芡欧鼠尾草(Salvia hispanica)的Remorin蛋白亲缘关系较近。构建pCAMBIA-SmREM1.2-GFP重组载体,利用烟草瞬时转化系统对SmREM1.2进行亚细胞定位分析,发现该蛋白定位于细胞膜上。通过实时荧光定量PCR分析发现SmREM1.2受盐胁迫诱导上调表达,推测其在盐胁迫下发挥重要作用。本研究结果可为后期深入探索SmREM1.2基因的功能及应用提供理论依据。

Remorin is a plantspecific oligomeric filamentous protein that is localized on cell membrane and mediates the formation of lipid rafts into plasma membrane microregions.It plays vital roles in plant response to abiotic and biotic stresses and immune regulation.Salvia miltiorrhiza Bunge is one of the bulk Chinese medicinal herbs and is widely used for the treatment of cerebrovascular and cardiovascular diseases.However,currently there are fewer reports about the function research of Remorin in S.miltiorrhiza.In this study,we cloned the S.miltiorrhiza Remorin gene SmREM1.2 based on the transcriptome and genome databases of S.miltiorrhiza,used bioinformatics methods to analyze its amino acid composition,conserved domains,phylogenetic evolution and subcellular localization,and also expression level under salt stress.The results indicated that the length of coding sequence(CDS)of SmREM1.2 was 585 bp,and it encoded 194 amino acids.SmREM1.2 protein was a weakly acidic and unstable hydrophilic protein with no signal peptide and transmembrane regions.It contained the conserved domains of Remorin_N and Remorin_C.Its secondary structure was mainly composed of alpha helices and irregullar curls.SmREM1.2 had the highly conserved coiledcoil domain at Cterminal region,and belonged to the typical REM protein.Phylogenetic analysis results showed that SmREM1.2 was very closely related to Salvia hispanica REM protein.Through constructing the pCAMBIASmREM1.2GFP vector and using the tobacco transient transformation system,the subcellular localization of SmREM1.2 was analyzed.It was found that SmREM1.2 was localized on membrane.The realtime quantitative PCR results showed that the expression of SmREM1.2 was upregulated by salt stress treatment,suggesting that this gene play an important role in plant salt stress response.All the results of this study could provide a theoretical basis for further exploration of function and application of SmREM1.2 gene.