Cloning and Expression Analysis of miR408 Precursor Sequences from Salvia miltiorrhiza
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摘要: MiR408是植物中一类高度保守的miRNA,其靶基因编码含铜蛋白,miR408的表达受植物生长发育和环境条件的显著影响。拟南芥中miR408在HY5-SPL7基因网络中起着关键作用,而HY5-SPL7基因网络可介导拟南芥对光照和铜离子的协调应答,进一步表明miR408在植物对环境的响应中发挥了核心作用。本研究基于丹参基因组Survey数据库,从中搜索并克隆得到366bp的含有稳定茎环结构的miR408前体序列及其上游723bp片段的启动子区,GenBank登录号分别为KU360384和KU360385,并将miR408的前体序列命名为Sm-MIR408。生物信息学分析结果显示:Sm-MIR408上游启动子序列与模式植物拟南芥Ath-MIR408、水稻Osa-MIR408启动子区具有许多相同的顺式作用元件,如G-box、CGTCA-motif、TGACG-motif、GTAC-motif等,而HSE、CATT-motif作用元件仅存在于丹参启动子区;miR408成熟序列在不同物种中具有很高的保守性;基于不同物种miR408前体序列构建的系统进化树显示,来源于单子叶植物的miR408前体序列聚为一支,来源于双子叶植物的miR408前体序列聚为另一支。实时定量PCR分析结果显示:Sm-MIR408在丹参的根、茎、叶、花中都有表达,其在花中的表达量最高,根中最低;Sm-MIR408的表达受茉莉酸甲酯和伤害的抑制,黑暗和持续光照也可抑制该基因的表达。Sm-MIR408基因的克隆与表达分析为今后研究丹参miR408的功能奠定了基础。Abstract: MiR408 is a highly conserved miRNA in plants and responds to copper availability by targeting genes encoding copper-containing proteins. Its expression is significantly affected by plant growth and development and environmental conditions. MiR408 is critical for the HY5-SPL7 gene network in Arabidopsis, and mediates a coordinated response to light and copper, illustrating its central role in the reaction of plants to the environment. Based on the genomic survey database of Salvia miltiorrhiza established in our lab, a 366 bp miR408 precursor sequence with stable stem loop structure was cloned and named Sm-MIR408. The 723 bp promoter region upstream of Sm-MIR408 was obtained by PCR. The GenBank accession numbers of Sm-MIR408 and the promoter sequence are KU360384 and KU360385, respectively. Bioinformatic analysis showed that the promoter region of Sm-MIR408 shared the same cis-acting elements as those of Ath-MIR408 in Arabidopsis thaliana and Osa-MIR408 in Oryza sativa, and included G-box, CGTCA-motif, TGACG-motif and GTAC-motif, whereas HSE and CATT-motif elements existed only in the Sm-MIR408 promoter region. Mature miR408 is highly conserved among different species. A phylogenetic tree of miR408 from different species showed that miR408 precursor sequences from monocotyledons were clustered on one branch and those from dicotyledons were clustered on another. Real-time quantitative PCR showed that the expression of Sm-MIR408 in the roots, stems, leaves and flowers of S. miltiorrhiza was the highest in the flowers and the lowest in the roots. Expression of Sm-MIR408 could be suppressed by methyl jasmonate, wounding, and continuous light or darkness. The cloning and expression analysis of Sm-MIR408 presented in this research has laid a foundation for studying the function of miR408 in S. miltiorrhiza in the future.
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Keywords:
- Salvia miltiorrhiza Bunge /
- MiR408 /
- Precursor sequence /
- Expression analysis
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