Selection and Validation of Optimal RT-qPCR Reference Genes for the Normalization of Gene Expression under Different Experimental Conditions in Lindera megaphylla

Abstract

Lindera megaphylla, a broad-leaved evergreen that is used as a landscape ornamental plant and medicinal plant, is an ecologically important and dominant tree species. However, little is known about the molecular mechanisms of its growth, development, and metabolism. The selection of suitable reference genes is critical for molecular biological analyses. To date, no research on reference genes as a foundation for gene expression analysis has been undertaken in L. megaphylla. In this study, 14 candidate genes were selected from the transcriptome database of L. megaphylla for RT-qPCR assay under different conditions. Results showed that helicase-15 and UBC28 were most stable in different tissues of seedlings and adult trees. For different leaf developmental stages, the best combination of reference genes was ACT7 and UBC36. UBC36 and TCTP were the best under cold treatment, while PAB2 and CYP20-2 were the best under heat treatment. Finally, a RT-qPCR assay of LmNAC83 and LmERF60 genes were used to further verify the reliability of selected reference genes above. This work is the first to select and evaluate the stability of reference genes for the normalization of gene expression analysis in L. megaphylla and will provide an important foundation for future genetic studies of this species.

Selection of Reference Genes for Expression Normalization by RT-qPCR in Dracocephalum moldavica L.

“…We identified 12 candidate reference genes by analyzing the annotations and FPKM values from a full-length and transcriptome dataset generated from various tissues and simulated drought treatment samples to assess the levels of gene expression. Liu et al obtained the candidate reference genes based on FPKM values greater than 50 [ 27 ]; Zhang et al did the same according to the variable coefficient of FPKM [ 44 ]. To screen more reference genes, we selected the FPKM with a numerical value and stable expression levels in all test samples.…”

“…Numerous studies have been conducted to screen the stability of reference genes in many plant species, including Schima superba [ 11 ], Ipomoea batatas [ 9 ], Stipagrostis pennata [ 10 ], Schima superba [ 15 ], Avena sativa [ 12 ], Vitis vinifera [ 16 ], Populus [ 17 ], Solanum nigrum [ 18 ], Cryptomeria fortune [ 19 ], Stellera chamaejasme [ 20 ], Dianthus broteri [ 21 ], Pyrus pyrifolia [ 22 ], Arachis hypogaea [ 23 ], and Nitraria tangutorum [ 24 ]. In plant gene expression studies, reference genes such as elongation factor-1α ( EF1α ), 18S rRNA , glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), Actin ( ACT ), S-adenosylmethionine synthase ( SAMS ), and tubulin ( TUB ) have commonly been employed for optimizing gene expression [ 9 , 25 , 26 , 27 ].…”

A Single Latent Plant Growth-Promoting Endophyte BH46 Enhances Houttuynia cordata Thunb. Yield and Quality

“…cordata leaves were analyzed by using quantitative reverse transcription PCR (qRT-PCR). Total RNA was extracted according to a previously described method H.…”

“…Total RNA was extracted according to a previously described method. 27 H. cordata leaves were ground to powder in liquid nitrogen, and total RNA was extracted using HiPure HP Plant RNA Mini Kit (Magen, Guangzhou, China) for quantification on a 7500 fluorescence quantitative PCR instrument. The relative expression of TR16816_c0_g1, TR477_c0_g1, TR2261_c0_g1, TR110415_c0_g1, and TR14326_c0_g1 was calculated using the 2 −ΔΔCT method.…”

Molecular Biology of Ornamental Plants

“…Continuous updates in technology and tools promote further studies of ornamental plants. Liu et al selected 14 candidate genes from the transcriptome database of Lindera megaphylla for additional qRT-PCRs under different conditions to further verify the reliability of selected reference genes above [15]. Cheng et al developed an efficient virus-induced gene silencing (VIGS) system using the leaf tip needle injection method, which could effectively silence genes in Lycoris chinensis, providing a powerful tool for gene function studies [16].…”