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United States Department of Agriculture

Agricultural Research Service

Research Project: Engineering Enzymatic Redirection of Natural Crop Oil Production to Industrial Oil Production Title: Characterization of reference gene expression in tung tree (Vernicia fordii)

Authors
item Cao, Heping
item Cao, Fangping -
item Klasson, K Thomas

Submitted to: Industrial Crops and Products
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 11, 2013
Publication Date: October 31, 2013
Citation: Cao, H., Cao, F., Klasson, K.T. 2013. Characterization of reference gene expression in tung tree (Vernicia fordii). Industrial Crops and Products. 60:248-255.

Interpretive Summary: Tung tree is a tropical plant with a very limited growing area in the southeastern United States. Tung tree produces large seeds containing about 50-60% oil (dry weight basis). Tung oil is readily oxidized due to the three unique conjugated double bonds in eleostearic acid. Thus, tung oil 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. A number of genes have been identified for tung oil biosynthesis. However, less is known about the expression patterns of many 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. In this study, we characterized the expression of three 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, glyceraldehyde 3 phosphate dehydrogenase and ubiquitin protein ligase was examined by TaqMan and SYBR Green qPCR using RNA from three trees, three tissues and 11 time events of developing seeds. The variations of the three mRNA levels were compared between two RNA extraction methods and two cDNA preparations in 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. These results suggest that Rpl19b and Ubl are preferable reference genes for both qPCR assays for quantitative gene expression analysis in tung trees. The assay development will help to identify key factors for tung oil biosynthesis and provide potential targets for genetic engineering value-added oil crops.

Technical 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 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. These results suggest that Rpl19b and Ubl are preferable reference genes for both TaqMan and SYBR Green qPCR assays for quantitative gene expression analysis in tung trees.

Last Modified: 10/24/2014
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