Cloning and expression analysis of pyruvate kinase gene in Dendrobium officinale
10.7501/j.issn.0253-2670.2014.07.018
- Author:
Lin ZHANG
1
Author Information
1. Shaanxi Provincial Key Laboratory for Chinese Medicine Basis and New Drugs Research, College of Pharmacy, Shaanxi University of Chinese Medicine
- Publication Type:Journal Article
- Keywords:
Bioinformatics;
Dendrobium officinale Kimura et Migo;
Expression analysis;
Gene cloning;
Pyruvate kinase
- From:
Chinese Traditional and Herbal Drugs
2014;45(7):990-995
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To clone the pyruvate kinase (PK) gene DoPK in a rare and endangered medicinal plant Dendrobium officinale, followed by bioinformatic analysis and to detect the expression in different organs. Methods: RT-PCR and RACE technologies were used to clone the full length cDNA of DoPK gene. The molecular characteristics such as physiochemical properties, conserved domain, and sub-cellular localization of the deduced DoPK protein were determined using a series of bioinformatic tools. The analyses of multiple alignment and phylogenetic tree were performed using DNASTAR 6.0 and MEGA 4.0 softwares, respectively. Real time quantitative PCR (RT-qPCR) was used for gene expression analysis in different organs. Results: The full length cDNA of DoPK was 1895 bp in length (GenBank accession KC178572) and encoded the protein of 511 amino acids with a molecular weight of 55040 and an isoelectric point (PI) of 7.00. Sequence analysis demonstrated that DoPK was similar to PK genes from Selaginella moellendorffii, Arabidopsis thaliana, Solanum tuberosum, and Vitis vinifera with the identities of 71%, 86%, 89%, and 91%, respectively. The deduced DoPK protein contained the conserved PK domain (21-497) and the active site (235-247). Phylogenetic analysis indicated that DoPK was grouped into the CYTOSOLIC-1 subfamily and was closely related to monocots. The transcription level of DoPK in the stems of D. officinale was the highest (2.29-fold higher than that in the leaves), followed by that in the roots (1.28-fold). Conclusion: The full length cDNA sequence in a CYOTOSLIC DoPK gene is identified, facilitating further functional analysis of the gene involving in the growth and development of D. officinale.
