Cloning and expression analysis of a novel alkaline/neutral invertase (DoNI2) gene in Dendrobium officinale
10.7501/j.issn.0253-2670.2018.15.027
- Author:
Xiao-Rong MIAO
1
Author Information
1. Agricultural College, Guangxi University
- Publication Type:Journal Article
- Keywords:
Alkaline/neutral invertase;
Dendrobium officinale Kimura et Migo;
Gene cloning;
Gene expression;
QRT-PCR
- From:
Chinese Traditional and Herbal Drugs
2018;49(15):3659-3666
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To clone novel member of alkaline/neutral invertase (NI) gene in a rare and endangered medicinal plant of Dendrobium officinale, conduct bioinformatic analysis and detect the quantitative expression in different organs. Methods: Primers were designed according to NI gene segment which was selected from leaf transcriptome sequencing results of D. officinale. The full-length cDNA of NI gene was cloned via homology-based cloning and rapid amplification of cDNA ends (RACE) approach. The physical and chemical properties, secondary structure and tertiary structure of NI protein were forecasted and analyzed using related software. The expression levels of NI gene in roots, stems, and leaves of D. officinale were detected using real-time PCR. Results: A novel gene encoding a NI protein was cloned from D. officinale. This gene (named as DoNI2, GenBank accession number: KY794404) had a total length of 2 397 bp with an open reading frame of 1 836 bp, and encoded a predicted polypeptide of 611 amino acids with a molecular weight of 69 050. Bioinformatics predicted that the isoelectric point of DoNI2 gene encoding protein was 6.38, the instability coefficient was 44.95, and the hydrophobic coefficient was −0.232. RT-PCR showed that DoNI2 gene expressed in all organs with highest expression level in stems and the lowest in roots. DoNI2 gene expression was significantly positively correlation with NI enzymatic activities at different growth years of D. officinale. Conclusion: The full length cDNA sequence in a mitochondrial DoNI2 gene was identified, facilitating future functional analysis of the gene involving in the regulation of sugar metabolism in D. officinale.
