Characterization of reference gene expression in tung tree (Vernicia fordii)

Abstract

Tung oil from tung tree (Vernicia fordii) is widely used as a drying ingredient in paints, varnishes, and other coatings and finishes. Recent research has focused on the understanding of the biosynthesis of oil in tung trees. Many oil biosynthetic genes have been identified in tung tree but little is known about the expression patterns of the genes in tung seeds. Quantitative real-time-PCR (qPCR) assays are widely used for gene expression analysis. One crucial task of qPCR assay design is to select stably expressed internal reference genes for data analysis. The objective of this study was to characterize the expression of potential reference genes in the tung tree to provide a sound basis for reliable and reproducible qPCR results. The expression of tung 60s ribosome protein L19 (Rpl19b), glyceraldehyde 3 phosphate dehydrogenase (Gapdh) and ubiquitin protein ligase (Ubl) was examined by TaqMan and SYBR Green qPCR using RNA from three trees, three tissues (seeds, leaves and flowers) and 11 time events of developing seeds. The variations of the three mRNA levels were compared between two RNA extraction methods, two cDNA preparations, and the same or different PCR plates. Overall results demonstrated that Rpl19b was the most stably expressed gene, followed closely by Ubl, and Gapdh was the worst among the three genes under optimized qPCR assay conditions. These results suggest that Rpl19b and Ubl are preferable reference genes for both TaqMan and SYBR Green qPCR assays. The development of these reference genes for quantitative gene expression analyses in tung trees should facilitate identifying target genes for genetic engineering industrial oils in oilseed crops.