Cloning and evaluation of reference genes for quantitative real-time PCR analysis inAmorphophallus

Abstract

Quantitative real-time reverse transcription PCR (RT-qPCR) has been widely used in the detection and quantification of gene expression levels because of its high accuracy, sensitivity, and reproducibility as well as its large dynamic range. However, the reliability and accuracy of RT-qPCR depends on accurate transcript normalization using stably expressed reference genes. Amorphophallus is a perennial plant with a high content of konjac glucomannan (KGM) in its corm. This crop has been used as a food source and as a traditional medicine for thousands of years. Without adequate knowledge of gene expression profiles, there has been no report of validated reference genes in Amorphophallus. In this study, nine genes that are usually used as reference genes in other crops were selected as candidate reference genes. These putative sequences of these genes Amorphophallus were cloned by the use of degenerate primers. The expression stability of each gene was assessed in different tissues and under two abiotic stresses (heat and waterlogging) in A. albus and A. konjac. Three distinct algorithms were used to evaluate the expression stability of the candidate reference genes. The results demonstrated that EF1-a, EIF4A, H3 and UBQ were the best reference genes under heat stress in Amorphophallus. Furthermore, EF1-a, EIF4A, TUB, and RP were the best reference genes in waterlogged conditions. By comparing different tissues from all samples, we determined that EF1-α, EIF4A, and CYP were stable in these sets. In addition, the suitability of these reference genes was confirmed by validating the expression of a gene encoding the small heat shock protein SHSP, which is related to heat stress in Amorphophallus. In sum, EF1-α and EIF4A were the two best reference genes for normalizing mRNA levels in different tissues and under various stress treatments, and we suggest using one of these genes in combination with 1 or 2 reference genes associated with different biological processes to normalize gene expression. Our results will provide researchers with appropriate reference genes for further gene expression quantification using RT-qPCR in Amorphophallus.

RNA‐seq‐based selection of reference genes for RT‐qPCR analysis of pitaya

“…We found that the two algorithms produced the same ranking of the reference genes according to the differences in the stability (Table 2 and Fig. Many studies revealed that using a combination of multiple top stable reference genes might improve the reliability of gene expression by RT-qPCR [20,41]. The NormFinder analysis showed that HuUBQ and HuUQT were the two top stable reference genes in all of the experimental samples, while HuACT (under salt stress, drought stress, and different cultivars) and Hu18S rRNA (under heat stress and different tissues) were the most unstable reference genes.…”

“…The expression stabilities of these genes were found to be different in different species as well as different experimental conditions in the same species [5,9,20,22]. The expression stabilities of these genes were found to be different in different species as well as different experimental conditions in the same species [5,9,20,22].…”

“…In perennial ryegrass, UBQ and UQT were the best reference genes under waterlogging and heat stress, respectively [9], while UBQ was validated as one of the least stable in tea plan and black locust [5,17]. EF1-a was one of the stable expression genes in perennial ryegrass, Amorphophallus, and Deschampsia antarctica plants [7,9,20]. EF1-a was one of the stable expression genes in perennial ryegrass, Amorphophallus, and Deschampsia antarctica plants [7,9,20].…”

“…Many reference genes have been identified in specific experimental conditions. For instance, elongation factor 1-alpha (EF1-a) was the best reference gene to normalize gene expression under heat stress, waterlogging, and across different tissues in Amorphophallus [20]. Ubiquitin (UBQ) and actin (ACT) were the most stable reference genes in different developing seeds and cultivars in the tung tree [21], while 18S ribosomal RNA (18S rRNA) was the optimum reference gene under the salinity and drought conditions in kenaf [22].…”

Identification and validation of reference genes for real-time qPCR normalization during Al-induced programmed cell death in peanut

“…Elongation factor EF1 has been reported to be the most stable gene in terms of expression in peanut under cold treatment (Chen et al 2011, Chi et al 2012, and in switch grass (Huang et al 2014a) and African oil palm (Xia et al 2014) under abiotic stresses. The expression stability of genes is related to the function they play in biological metabolic pathways (Wang et al 2017). Ubiquitin is a small protein existing in all eukaryotes with a conserved sequence and structure, which plays important roles in controlling cellular signal transduction (Zhang 2011).…”