Screening and validation of reference genes for real-time fluorescence quantitative PCR in wax apple

Abstract：【Objective】Normalizing through reference genes can make more accurate and reliable results from reverse transcription real-time quantitative polymerase chain reaction (q PCR) . Recent studies have shown that no single housekeeping gene is universal for all experiments. Thus, suitable reference genes should be the first step of q PCR analysis. Up to now, there have been no related studies about reference genes adaptable to wax apple [Syzygium samarangense (BI.) Merr. et Perry]. The fruit quality instability and chilling injure in winter were two important problems in production of wax apple. In order to identify stable and reliable expressive genes of wax apple for studying the molecular mechanism of fruit quality formation and low temperature stress responses, seven candidate reference genes (ACT-7, GAPDH, UBQ, α-TUB, CYP20-1, EF-2, APRT-5) were evaluated for transcript normalization.【Methods】Three varieties of wax apple ('Tub Ting Jiang''Black Pearl'and'Feicui') with different peel colors were used in this study. Fruits were harvested on 10, 20, 30, 40, and 50 days after flowering (DAF) respectively from three trees of each variety in 2016. Twenty representative fruits were sampled from each tree in each developmental stage. Skins were separated from the pulp and immediately frozen in the liquid nitrogen and kept at-80 ℃ until use. Annual cutting seedlings of three wax apple varieties were placed in artificial climate boxes for temperature treatments by 24 ℃, 4 ℃, 1 ℃, and-2 ℃ without light for 3 hours. Each treatment used 1 one year old tree propagated by cutting and repeated threetimes. The young leaves were collected for RNA isolation. Total RNA was extracted from each sample using a Plant Total RNA Extraction Kit (Bio Teke) . The integrity of RNA was assessed by 1.5% (ω) agarose gel electrophoresis. The concentrations of RNA were estimated with the Nanodrop2000 C spectrophotometer (Thermo) . The RNA in which the mean A260/A280 ratio was between 1.8-2.0 with perfect integrity was used for reverse transcription. The first strand c DNA was synthesized using Prime Scrip 1 st Strand c DNA Synthesis Kit (Ta Ka Ra) according to the manufacturer's instructions. The sequences of seven reference genes and one validation gene (flavanone-3-hydroxylase gene, F3 H) were obtained from our wax apple RNA-seq transcriptome dataset and compared with the Arabidopsis Genome Initiative Protein Database using BLASTX. The qRT-PCR reactions were performed in 96-well plates with a Bio-Rad real-time PCR system, the reaction contained 10 μL SYBR®Premix, 4 μL c DNA, 0.4 μL of each primer, 0.4 μL Rox in a final volume of 25 μL. The qRT-PCR conditions were as follows: 95 ℃for 30 s, followed by 40 cycles of 5 s at 95 ℃, 30 s at 58 ℃. After the cycles, the melting curves were generated to verify the specificity of primers. The amplification efficiency of the primers was estimated using the PCR program. The expression levels of the candidate reference genes were determined by Ct values, and relative expression levels were imported into geNorm, NormFinder, BestKeeper and RefFinder to analyze the gene expression stability.【Results】The ultraviolet spectrophotometer detection showed that the A260/A280 and A260/A230 values of the RNA samples were between 1.8 and 2.0. Obvious bands of 28 S, 18 S and 5 S r RNA were obtained by agarose gel electrophoresis, indicating high quality RNA for c DNA synthesis. The Ct values of seven candidate reference genes ranged from 18.88 to 31.59. The Ct values of seven genes in the leaves were between 18.88 and 29.87, and those in the flesh and peel were between 25.44 and 31.53, indicating that the seven genes in the leaves had higher expression abundance than those in the fruits. In addition, the gene of EF-2 presented higher expression levels than other genes, and α-TUB showed the lowest expression level. Some reports suggested that different analysis software would result in different validation results, and our results also showed that different suitable reference genes could be selected by four analysis approaches in this paper. The results of the geNorm analysis were similar to those of NormFinder and RefFinder, the most stable reference genes were ACT-7, α-TUB and CYP20-1 during the flesh and peel developmental process and under low temperature stress, respectively. Different from the above, the results of BestKeeper analysis showed that UBQ was the most stable reference gene under various experiment conditions. Meanwhile, the expression of flavanone-3-hydroxylase gene in the pathway of wax apple anthocyanin biosynthesis was analyzed, and the result indicated that the variation tendency of F3 H was exactly consistent using α-TUB and UBQ as reference genes.【Conclusion】Different suitable reference genes for wax apple should be selected case by case, and the most stable reference genes were different under various experiment conditions. ACT-7, GAPDH, UBQ and α-TUB housekeeping genes could be used as superior reference genes for normalization of gene expression measures for wax apple flesh development, α-TUB and UBQ for peel development and CYP20-1 and UBQ in leaves for low temperature stress.