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Pak. J. Bot., 47(4): 1265-1270, 2015.

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cDNA cloning and expression analyses of the isoflavone reductase-like gene of Dendrobium officinale

XIONG QIAN^1,2, SHAO-ZHONG XU^1,2, CHANG-LING ZHAO^1,2, HENG-LING MENG³ AND GUO-SONG WEN^1,2*

Abstract: The full length of the isoflavone reductase-like gene (IRL) cDNA of Dendrobium officinale was cloned by using reverse transcription (RT) PCR combined with cDNA library, the IRL function was identified by Bioinformatics and prokaryotic expression analyses, and the IRL expression levels in the organs and tissues of D. officinale plants with different ages were determined by using real-time quantitative PCR (RT-qPCR). The results indicated that the full length of the cDNA of D. officinale IRL, DoIRL, was 1238 bp (accession no. KJ661023). Its open reading frame (ORF) was 930 bp which encoded 309 amino acids with a predicted molecular mass of 34 kDa, the 5′ untranslated region (UTR) was 61 bp and the 3′ UTR containing a poly (A) tail was 247 bp. The deduced amino acid sequence of DoIRL, DoIRL, was forecast to contain a NAD(P)H-binding motif (GGTGYIG) in the N-terminal region, two conserved N-glycosylation sites, a conserved nitrogen metabolite repression regulator (NmrA) domain and a phenylcoumaran benzylic ether reductase (PCBER) domain, to hold the nearest phylogenetic relationship with the PCBER of Striga asiatica, and to share both 73% identity with the isoflavone reductases-like (IRLs) of Cucumis sativus and Striga asiatica. In Escherichia coli ‘BL21’ cells, the DoIRL cDNA expression produced a protein band holding the predicted molecular mass of 34 kDa. DoIRL expressed in all organs and tissues of D. officinale plants with different ages at comparatively low levels, and the expression level in the leaves of the two-year-old plants was the highest.

Key words: cDNA, Cloning, Reductase-like gene, Dendrobium officinale, RT-qPCR.

¹College of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming 650201, China

²Institute of the Improvement and Utilization of Characteristic Resource Plants, Yunnan Agricultural University, Kunming 650201, China

³Key Laboratory of High Quality Crops Cultivation and Safety Control, Honghe University, Mengzi 661100, China

* Corresponding author’s e-mail: wengs@163.com