Cloning and tissue-specific expression analysis of conserved fragments of DXR gene from Angelica sinensis / 中草药

ABSTRACT

Objective: To clone the conserved cDNA fragment of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) gene from Angelica sinensis and to analyze its tissue-specific expression in roots, stems, and leaves. Methods: A pair of degenerate primers were designed according to the conservative sequences of the cloned DXR from other plant species. The total RNA from the leaves of A. sinensis was as template, DXR fragment was obtained by reverse transcription polymerase chain reaction (RT-PCR), connected to pGEM-T Easy vector then transformed into Escherichia coli DH5α. The positive clone identified by PCR was sequenced. Taking Actin of A. sinensis as a reference gene, SYBR Green quantitative RT-PCR was used to detect the relative expression levels of DXR different tissues of A. sinensis. Results: The DXR fragment of A. sinensis containing 564 bp encoding 187 amino acids was registered in Genbank (No. KJ000259). The sequence identity analysis suggested that both the obtained nucleotide sequence and its corresponding amino acid sequence shared more than 80% of homology with GenBank DXRs from Gossypium barbadense and other five higher plant species. DXR was expressed at the highest level in the leaves, and the relative expression levels in the leaves were 2.5 and 3.2 times relative to the stems and roots, respectively (P<0.01). Conclusion: A novel DXR fragment is cloned from A. sinensis and its tissue-specific expression in A. sinensis is investigated. This work might establish an experimental basis for the effective application of DXR in the future.