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作 者 ：刘 冉,周立敬,周宜君*,高 飞,马亭亭,隋 欣,刘 楠

期 刊 ：西北植物学报 2012年 4期 页码：665~669

关键词：盐芥；miRNAs；茎环的反转录PCR；盐胁迫；

Keywords:Thellungiella, microRNAs, stem-loop RT-PCR, salt stress,

摘 要 ：以盐生植物盐芥为实验材料，选择经过Solexa测序筛选的盐芥tsa-miR172a和tsa-miR398b为目标基因，采用茎环的反转录PCR（stem-loop RT-PCR）方法分析其在盐芥根中的耐盐表达模式，以探讨盐芥miRNAs的stem-loop RT-PCR验证体系。结果显示，经过300 mmol·L^-1 NaCl处理72 h后，与对照相比，盐芥根中的tsa-miR172a上调表达，tsa-miR398b下调表达。用stem-loop RT-PCR方法进行tsa-miR172a和tsa-miR398b扩增，对其电泳图进行光密度分析结果显示，盐胁迫处理与对照的比值分别为1.8和0.55，Solexa测序结果分别为2.00和0.44，说明两种方法所得结果基本一致。表明该研究建立了盐芥miRNAs的stem-loop RT-PCR验证体系： 每个miRNA设计3个引物（miRNA stem-loop引物、miRNA正向引物和miRNA通用反向引物）；扩增条件为94℃ 2 min，94℃ 15 s，55℃ 45 s，23个循环。

Abstract:The expressions of tsa-miR172a and tsa-miR398b in roots of Thellungiella halophila (T.salsuginea),which were screened via Solexa sequencing,were studied by stem-loop RT-PCR methodology.The results showed that tsa-miR172a was up-expressing and tsa-miR398b was down-expressing under the treatment of NaCl (300 mmol·L^-1,72 h).With the stem-loop RT-PCR,tsa-miR172a and tsa-miR398b were amplified and the optical densities of electrophores were analyzed specifically.The ratios of NaCl stress to control were 1.8 and 0.55,which were roughly consistent with the results 2.00 and 0.44 obtained by Solexa.Our study established an effective stem-loop RT-PCR system:three primers,namely miRNA stem-loop primer,miRNA forward primer and miRNA universal reverse primer,were designed for each miRNA.The reactions were incubated at 94℃ for 2 min,followed by 23 cycles of 94℃ for 15 s and 55℃ for 45 s.The stem-loop RT-PCR system to identify miRNAs in Thellungiella was discussed.

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