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目的克隆太子参肌动蛋白(Actin)基因片段并进行序列分析。方法利用植物Actin基因的保守序列设计简并引物,通过RT-PCR和抑制PCR扩增太子参Actin基因核心片段。利用半定量RT-PCR分析太子参Actin基因在不同种源、不同器官、不同生长发育时期的表达情况。结果通过RT-PCR获得3条太子参Actin基因的核心片段,长度均为760 bp,依次命名为PhACT1、PhACT2、PhACT3。通过抑制PCR及序列拼接后3条核心片段依次延长至1 008、1 008、975 bp,分别编码336、336、325个氨基酸残基。半定量RT-PCR分析表明PhACT2和PhACT3基因在不同种源、不同器官、不同生长发育时期的表达量基本恒定,PhACT1基因存在一定的差异。结论首次从太子参中克隆得到3条太子参Actin基因序列,并确定PhACT2基因适合作为太子参功能基因表达分析的内参基因。
Objective To clone the Actin gene and analyze the sequence. Methods Degenerate primers were designed based on the conserved sequence of plant Actin gene. The core fragment of Actin gene was amplified by RT-PCR and PCR. Semi-quantitative RT-PCR was used to analyze the expression of Actinomyces heterophylla in different provenances, organs and different growth stages. Results The core fragments of three Actin genes were obtained by RT-PCR. The lengths of the two fragments were 760 bp, named PhACT1, PhACT2 and PhACT3 respectively. The 3 core fragments were successively extended to 1 008, 1 008 and 975 bp by inhibiting PCR and sequence splicing, respectively encoding 336,336,325 amino acid residues. Semi-quantitative RT-PCR analysis showed that the expression levels of PhACT2 and PhACT3 genes were basically constant in different provenances, organs and different growth stages, and there was a certain difference in PhACT1 gene. Conclusion For the first time, three Actin gene sequences were cloned from Pseudostellaria heterophylla, and PhACT2 gene was identified as an internal control gene for gene expression analysis of Pseudostellaria heterophylla.