Evaluation of Reference Genes for Normalizing RT-qPCR and Analysis of the Expression Patterns of WRKY1 Transcription Factor and Rhynchophylline Biosynthesis-Related Genes in Uncaria rhynchophylla

Abstract

Uncaria rhynchophylla (Miq.) Miq. ex Havil, a traditional medicinal herb, is enriched with several pharmacologically active terpenoid indole alkaloids (TIAs). At present, no method has been reported that can comprehensively select and evaluate the appropriate reference genes for gene expression analysis, especially the transcription factors and key enzyme genes involved in the biosynthesis pathway of TIAs in U. rhynchophylla. Reverse transcription quantitative PCR (RT-qPCR) is currently the most common method for detecting gene expression levels due to its high sensitivity, specificity, reproducibility, and ease of use. However, this methodology is dependent on selecting an optimal reference gene to accurately normalize the RT-qPCR results. Ten candidate reference genes, which are homologues of genes used in other plant species and are common reference genes, were used to evaluate the expression stability under three stress-related experimental treatments (methyl jasmonate, ethylene, and low temperature) using multiple stability analysis methodologies. The results showed that, among the candidate reference genes, S-adenosylmethionine decarboxylase (SAM) exhibited a higher expression stability under the experimental conditions tested. Using SAM as a reference gene, the expression profiles of 14 genes for key TIA enzymes and a WRKY1 transcription factor were examined under three experimental stress treatments that affect the accumulation of TIAs in U. rhynchophylla. The expression pattern of WRKY1 was similar to that of tryptophan decarboxylase (TDC) under ETH treatment. This research is the first to report the stability of reference genes in U. rhynchophylla and provides an important foundation for future gene expression analyses in U. rhynchophylla. The RT-qPCR results indicate that the expression of WRKY1 is similar to that of TDC under ETH treatment. It may coordinate the expression of TDC, providing a possible method to enhance alkaloid production in the future through synthetic biology.

Genome-Wide Mining of CULLIN E3 Ubiquitin Ligase Genes from Uncaria rhynchophylla

“…The total RNAs from U. rhynchophylla leaves were isolated according to the manufacturer’s instructions via the SteadyPure Plant RNA Extraction Kit (Accurate Biology, Hunan, China). The primers used for qRT-PCR Specific primer pairs designed using Beacon Designer 7.0 software are listed in Supplementary Table S3 ; the primers of key enzyme genes involved in TIA biosynthesis pathway have been reported previously with the SAM gene as the internal reference gene [ 62 ]. The qRT-PCR was performed using 2× SYBR Green Pro Taq HS premix (Vazyme, Shanghai, China) and reaction system and procedure have been previously reported [ 63 ], using the following protocol: 95 °C for 3.00 min, 1 cycle; 95 °C for 15 s and 60 °C for 30 s, 40 cycles.…”

“…, which were phytohormone (89) and abiotic and biotic stress responsive(62) were identified (Figure4). The first category relates to plant growth and development, including light-responsive elements (Box4 (26); G-box (17); GT1-motif (26); GATA-motif (6); ATCTmotif (3); I-box (5); AT1-motif (2) et al; meristem-associated (CAT-box (9); O 2 -site (5); circadian and endosperm-expressing GCN4 motif (4).…”