Reference Gene Screening for Real-time Quantitative PCR in Red Pear (Pyrus pyrifolia)
ZHANG Xue1*, WANG Li1, QU Fei2, YANG Sheng-Jun3
1 Fruit Science Institute, Guizhou Academy of Agriculture Sciences, Guiyang 550006, China; 2 College of Agriculture, Guizhou University, Guiyang 550025, China; 3 Horticulture Institute, Guizhou Academy of Agriculture Sciences, Guiyang 550006, China
Abstract Synthesis and accumulation of cyanine nucleoside in Red pear (Pyrus pyrifolia) peel are key factors influencing red pear fruit coloring and biosynthesis regulated by enzymatic reaction genes, screening of stable and reliable reference genes has important significance to research the key genes and their regulatory mechanism in red pear fruit coloring. In order to select reference genes for qRT-PCR analysis in different growth period (young fruit period,slow growth period, fast growth period, mature period) and different shading conditions (natural light, partial shading, complete shading) in different varieties (Zaobaimi, Zhongshu32, Yunhongli No.1) of red pear fruit, 6 common house-keeping genes were chosen as candidate for internal gene which included β-actin (ACT), 18S ribosomal RNA (18S rRNA), glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH), histone (His), elongation factor 1α (EF-1α), and α-tubulin (TUB). The qRT-PCR amplification efficiency, the stability parameters and the relative expression in different samples of internal reference genes were analyzed. The BIO-RAD CFX Manager v2.0 software was used to analyze and compare the stability parameter index such as cycle threshold (Ct), average expression coefficient (M) and variation coefficient (CV). The results showed different expression stability in different candidate genes, and EF-1α and His showed stable expression trend in different samples from different varieties, growth stages, and processing conditions. Analysis of relative expression of reference genes in different samples showed that the expression of EF-1α and His in different samples were relatively stable. As a result, EF-1α and His could be used as reference gene for qRT-PCR analysis in red pear peel tissues which might standardize genetic selection for relative quantitation about coloring related gene expression analysis in red pear.
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