为克隆扁果枸杞(Lycium barbarum Bianguo)肌动蛋白基因作为基因表达模式分析的内参基因,根据几种茄科植物肌动蛋白氨基酸序列保守区设计引物,以扁果枸杞3周龄叶总RNA 为模板,采用RT-PCR 的方法扩增肌动蛋白基因片段并连接到pMD18-T载体上,阳性克隆经PCR 检测后测序。结果表明,该基因片段长598 bp,编码198个氨基酸,与枸杞(Lycium chinense)核苷酸序列的相似度和同源性分别达97%和100%,说明是肌动蛋白基因片段,命名为LbACT。荧光定量PCR分析表明,盐处理下LbACT基因在各器官中的Ct值稳定,可作为内参基因用于研究扁果枸杞功能基因的表达模式分析。
The gene encoding actin was cloned from Lycium barbarum Bianguo and checked to be a potential reference gene to analyze the expression levels of other genes. The primers were designed based on conserved sequences of actin genes from other Solanaceae plants. The total RNA isolated from leaves of Lycium barbarum Bianguo was used as a template for reverse transcription-polymerase chain reaction (RT-PCR). The nucleotide fragment was amplified and cloned into pMD18-T. The positive clones were identified by PCR, followed by sequencing. The results revealed that the amplified fragment, named as LbACT, contained 598 bp, encoded 198 amino acid residues. The nucleotide sequence was 97% similar to that of Lycium chinense actin gene. Meanwhile, fluorescent quantitative PCR showed that salt-treated LbACT was stable in all organs. Therefore, LbACT could be used as a reference gene to analyze the expressions of functional genes of Lycium barbarum Bianguo.
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