Molecular cloning and expression analysis of two genes encoding F family ATP-binding cassette transporters in Dendrobium officinale / 中草药

ABSTRACT

Objective: To clone and characterize the F family ATP-binding cassette (ABC) transporter genes in Dendrobium officinale. Methods: RACE was used to isolate ABC transporter genes from the leaf cDNA of D. officinale. Characteristics including molecular weight, theoretical pI (isoelectric point), conserved domain, transmembrane structure, signal peptide, and subcellular localization of the deduced protein were analyzed by serials of bioinformatics algorithms. The analyses of multiple alignment and phylogenetic tree were respectively performed by DNASTAR and MEGA6. Tissue specific expression patterns were determined by real-time quantitative PCR (qPCR) analyses. Results: Two full length genes DoABCF1 and DoABCF2 (GenBank accessions KU160474 and KU160475), 2 104 and 2 193 bp in length, respectively, were obtained. DoABCF1 was deduced to a 600 aa (amino acid) protein with a molecular weight of 67 030 and pI of 6.20, while DoABCF2 encoded a 659 aa protein with a molecular weight of 74 140 and pI of 5.71. The two deduced DoABCF1 and DoABCF2 proteins both had two conserved ABC domains (74-314 and 385-600 for the former, 65-323 and 392-607 for the latter) and several functional motifs. The proteins did not contain any signal peptide or transmembrane domain, and were predicted to locate in the chloroplast. The two genes were highly identical to the plant F family ABC transporter genes with more than 80% similarity, and were mostly close to monocots ABC F family members from maize and rice. DoABCF1 and DoABCF2 showed different expression among the three organs and both had relatively highest expression levels in the leaves, whereas no significant difference could be observed in the roots and stems. Taken the stem as the calibrator sample, DoABCF1 transcripts were 1.74 fold over that in the stems and DoABCF1 transcripts were 3.44 fold, respectively. Conclusion: Two F family DoABCF1 and DoABCF2 genes with full length cDNAs are successfully cloned in this study. The highest expression levels of the two ABC genes in the leaves of D. officinale suggest that they should play an important role during the growth and development of D. officinale.