Cloning and Analysis of Gene Fragments Encoding C4H and CAD from Miscanthus sinensis
College of Agriculture, Henan University of Science and Technology, Luoyang, 471003,China;
Plant Gene and Trait, 2013, Vol. 4, No. 6 doi: 10.5376/pgt.2013.04.0006
Received: 17 Jul., 2013 Accepted: 19 Jul., 2013 Published: 23 Jul., 2013
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Preferred citation for this article:
Fan and Li, 2013, Cloning and Analysis of Gene Fragments Encoding C4H and CAD from Miscanthus sinensis, Plant Gene and Trait, Vol.4, No.6 30-36 (doi: 10.5376/pgt.2013.04.0006)
Based on cDNA sequences encoding Cinnamate-4-hydroxylase (C4H) and Cinnamoyl alcohol dehydrogenase (CAD) isolated from several monocots reported in GenBank, two pairs of PCR primers were designed with Primer Premier 5.0 software. The total RNA was extracted from Miscanthus sinensis according to CTAB-LiCl method and then cDNA was synthesized by reverse transcription. PCR products, which were named as MsC4H and MsCAD, were successfully obtained by reverse transcription polymerase chain reaction (RT-PCR) and then cloned into vector pMD18-T. The positive clones identified by plasmid PCR were sequenced. The sequencing results revealed that MsC4H and MsCAD contain 305 and 269 base pairs, which encode 101 and 51 amino acids, respectively. BLAST analysis showed that the cloned nucleotide sequences and their corresponding amino acids are highly homologous to many C4H and CAD genes and their encoding proteins from different plant species. The sequences of MsC4H and MsCAD had been submitted to Genbank database, whose accession numbers are JQ598686 and JQ598683, respectively.
Miscanthus sinensis; C4H; CAD, Cloning; Sequence analysis