Abstract
Quantitative real-time PCR (RT-qPCR) has become an accurate and widely used technique to analyze expression levels of selected genes. It is very necessary to select appropriate reference genes for gene expression normalization. In the present study, we assessed the expression stability of 11 reference genes including eight traditional housekeeping genes and three novel genes in different tissues/organs and developing seeds from four cultivars of tung tree. All 11 reference genes showed a wide range of Ct values in all samples, indicating that they differently expressed. Three softwares – geNorm, NormFinder and BestKeeper – were used to determine the stability of these references except for ALB (2S albumin), which presented a little divergence. The results from the three softwares showed that ACT7 (Actin7a), UBQ (Ubiquitin), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and EF1α (elongation factor 1-α) were the most stable reference genes across all of the tested tung samples and tung developing seeds, while ALB (2S albumin) was unsuitable as internal controls. ACT7, EF1β (elongation factor1-beta), GAPDH and TEF1 (transcription elongation factor 1) were the top four choices for different tissues/organs whereas LCR69 did not favor normalization of RT-qPCR in these tissues/organs. Meanwhile, the expression profiles of FAD2 and FADX were realized using stable reference genes. The relative quantification of the FAD2 and FADX genes varied according to the internal controls and the number of internal controls. The results further proved the importance of the choice of reference genes in the tung tree. These stable reference genes will be employed in normalization and quantification of transcript levels in future expression studies of tung genes.
Highlights
Tung tree (Vernicia fordii Hemsl.), a subtropical round-crowned deciduous tree, belongs to a species of the genus Vernicia in the spurge (Euphorbiaceae) family
The construction of cDNA library of tung seeds and the release of expressed sequence tag (EST) databases have greatly promoted the study of genes involved in fatty acids synthesis such as delta-12 fatty acid desaturase (FAD2), delta 12 fatty acid conjugase (FADX), diacylglycerol acyltransferase 1 (DGAT1) and diacylglycerol acyltransferase 2 (DGAT2) [6,7]
A total of 11 reference genes from the tung tree kernel uncut cDNA library were selected as candidates for normalization of gene expression measures
Summary
Tung tree (Vernicia fordii Hemsl.), a subtropical round-crowned deciduous tree, belongs to a species of the genus Vernicia in the spurge (Euphorbiaceae) family. Tung oil extracted from seeds is considered to be one of the high-value industrial oils [1], used widely in production of cloth, shoes, waterproofing masonry, clothing, paper, and biodiesel [1,2]. Following the development and maturation of tung tree seeds, the content of fatty acids gradually increases. The construction of cDNA library of tung seeds and the release of expressed sequence tag (EST) databases have greatly promoted the study of genes involved in fatty acids synthesis such as delta-12 fatty acid desaturase (FAD2), delta 12 fatty acid conjugase (FADX), diacylglycerol acyltransferase 1 (DGAT1) and diacylglycerol acyltransferase 2 (DGAT2) [6,7]. The understanding of expression patterns of some key genes will help elucidate the mechanism involved in fatty acids synthesis of tung seeds
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