太子参3个肌动蛋白基因片段的克隆与序列分析

Cloning and sequence analysis of three Actin gene fragments from Pseudostellaria heterophylla

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 Cloning and sequence analysis of the cDNA fragments encoding Actin gene from Pseudostellaria heterophylla. Methods Degenerate primers were designed based on the conservated sequences of the cloned Actin gene from other plant species. The core fragments ofActin gene were obtained by reverse transcription polymerase chain reaction （RT-PCR） and the flanking fragments were cloned by using suppression PCR. The expression profiles of Actin gene in different cultivated provenances, organs, and development stages were analyzed by semiquantitative PCR. Results The three Actin genes obtained were designated PhACT I, PhACT2, and PhACT3, which were extended to 1 008, 1 008, and 975 bp through suppression PCIL encoding 336, 336, and 325 amino acids, respecifively. The semiquantitative PCR analysis showed that PhACT2 and PhACT3 had a stable expression except PhACT1. Conclusion Three Actin genes are cloned and the PhACT2 could be used as an internal standard gene for the expression analysis of the functional genes in P. heterophylla.