Reference genes are used for RT-qPCR data normalization. The expression stability of reference genes can directly influence the accuracy of RT-qPCR, and reference genes should sustain stable expression levels in various tissues and under distinct experimental conditions [7, 38]. To our knowledge, the expression levels of genes vary with changes in tissue, species and experimental conditions [11]. Therefore, a gene must be strictly screened before being used as a reference gene to ensure the accuracy of RT-qPCR analysis. In addition, functional genes of P. edulis have not been well studied, especially by relative quantitative analysis of functional genes, and research on reference genes in P. edulis has not been reported. [1, 2]. To enhance the reliability of functional gene expression research, we systematically selected reliable internal control genes for RT-qPCR data normalization in multiple tissue samples and cold-treated samples of P. edulis.
In this work, we selected 10 candidate reference genes, namely, 50S, Ts, UBQ, OUT, OmpH, EF1, 18S, eIF5A, HIS, and Liom, and we used RT-qPCR to obtain their Ct values in 60 samples. Through analysis based on Ct values, we found that 18S had the highest expression level (5.99 Ct) and the smallest expression variation (1.94 Ct), while OmpH had the lowest expression level (26.75 Ct) and the largest expression variation (12.07 Ct) (Fig. 2). However, we could not use 18S a reference gene due to its low expression variation. According to previous studies, genes with low expression variation may not be suitable for data normalization, and reference genes must be evaluated comprehensively by using different tools [39, 40]. For example, research conducted on rice and D. longan showed that 18S cannot be used as an internal control gene [35, 36]. In particular, in research conducted on Bixa orellana, although 18S had the highest expression level and the smallest expression variation, further analysis results showed that 18S was the most unstable gene in multiple tissues and at different seed development stages [8]. This result indicated that Ct values of candidate reference genes were not enough to determine the expression stability.
Through further analysis by geNorm, NormFinder, and BestKeeper, Ts and EF1 were found to be ideal for data normalization in all samples (Table 3). The protein encoded by Ts can catalyze the last step of threonine biosynthesis, and there are many reports about its structure, expression and function [41, 42]. However, there are few reports about Ts as an internal control gene. EF1 is stably expressed in not only P. edulis, but also other plants. For example, EF1 was used as an internal control gene in Ilex paraguariensis leaves and potato (Solanum tuberosum L.) under abiotic stress [43, 44]. To our knowledge, the expression level of reference genes may change with changes in species, experimental conditions, and tissue types. For example, although EF1 was suitable for data normalization in P. edulis, potato under abiotic stress, drought-treated leaves of I. paraguariensis, and Betula luminifera, this gene was unsuitable for data normalization in Camellia sinensis under metal stress and in leaves of Camellia sinensis [38, 43–46]. Overall, Ts and EF1 have the highest expression stability, but if we classify all samples into different subsets, there may be other genes in each subset with higher expression stability than Ts and EF1.
To determine whether there were other genes that had higher expression stability than Ts and EF1 in different sample subsets, we classified the 60 samples into two subsets, namely, tissue samples and cold-treated samples. In previous studies on reference gene selection in different species, we found that the expression patterns of candidate reference genes may differ between nonstress and abiotic stress conditions [23, 47]. Under nonstress conditions, HIS and EF1 was the best combination for data normalization in tissue samples (Table 3). In L. multiflorum, HIS also showed a stable expression pattern under acidic aluminum stress and heavy metal stress, while it could not be selected as an internal control gene in Populus tomentosa due to its poor expression stability [26, 48]. Under cold stress conditions, UBQ and EF1 were sufficient for data normalization in P. edulis (Table 3). UBQ was also used as a reference gene in cotyledons of Cunninghamia lanceolata [11]. However, this gene was unsuitable for data normalization in Bursaphelenchus mucronatus and lettuce (Lactuca sativa) [49, 50]. Compared with Ts, HIS had higher expression stability in tissue samples and UBQ had higher expression stability in cold-treated samples. This finding indicates that HIS and UBQ can be used as internal control genes in only specific samples and experimental conditions. Moreover, 18S and Liom were unstably expressed in this study, and 18S also showed unstable expression patterns in amaranth, B. orellana and Stellera chamaejasme (Fig. 3, Fig. 5, Table 2) [8, 23, 51]. However, 18S could be used for data normalization in Bursaphelenchus mucronatus and C. sinensis under metal stress [45, 49].
As a nutritious tropical fruit, P. edulis is widely cultivated in southern China [2]. However, cold resistance has been an important factor limiting the expansion of the P. edulis cultivation area. Our laboratory discovered a cold-resistant variety, Pingtang 1, which is used as sample material for the study of cold resistance mechanisms. Our previous research has shown that the ICE1-CBF-COR pathway plays a crucial role in the cold tolerance of P. edulis [2]. ICE1 is a transcription factor that can regulate the expression of the downstream CBF gene, thereby improving plant resistance to cold stress [37]. To validate the reference genes that we selected, we used ICE1 as a target gene and Ts and EF1 as internal control genes, to investigate the expression level of ICE1 in cold-treated leaves of P. edulis. We found that the expression level of ICE1 increased with increasing cold-treatment times (Fig. 6). In previous studies, increased expression of ICE1 enhanced plant resistance to cold stress [37, 52]. Therefore, the increase in ICE1 expression may enhance the adaptability of P. edulis to cold stress.
Our work will facilitate the study of functional gene expression levels in P. edulis. We selected stable and reliable internal control genes and applied these genes in RT-qPCR data normalization in different tissue samples and under cold stress conditions in P. edulis.