Cloning and Expression Analysis of Actin Gene Fragment from Dioscorea opposite

doi:10.13560/j.cnki.biotech.bull.1985.2016.07.011

Abstract

Abstract: The cloning and expression analysis of Actin gene fragment from Dioscorea opposite was studied in order to provide the foundation for studying its function and other genes expression and regulation in yam growth and development. A pair of degenerate primers were designed based on the conserved sequences of the published Actin genes of other plants in GenBank. The cDNA fragment of 1 Actin gene was isolated from yam tuber by RT-PCR and designated as DoActin. It’s length was 1 091 bp encoding a protein with 357 amino acids residues,and was deposited in GenBank（Accession number：KU669295）. Homologous alignment according to the nucleotide and protein databases showed that it shared over 83% nucleotide sequence similarity and over 97% amino acid sequence similarity withActins in other plants. The phylogenetic tree reconstructed on the base of amino acid sequences suggested that the DoActin had the nearest relationship with Phoenix dactylifera Actin-2 and Gossypium arboreum Actin-7. RT-PCR results revealed that the expression levels ofDoActin in different organs such as leaf,aboveground stem and underground tuber,as well as tubers and leaves in different developmental periods were relatively stable,indicating that DoActin can be used as an appropriate reference gene for D. opposite.

Key words: Dioscorea opposite, Actin gene, cloning, expression analysis, sequence analysis

GONG Ming-xia, ZHOU Yun-yi, WANG Ai-qin, LUO Hai-ling, HE Long-fei. Cloning and Expression Analysis of Actin Gene Fragment from Dioscorea opposite[J]. Biotechnology Bulletin, 2016, 32(7): 73-80.

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