Toona ciliata Roem. belongs to the Meliaceae family, which is widely distributed in China, Australia and India. Because of its straight trunks and russet wood, T. ciliata has the title of "Chinese Mahogany" [1]. But due to environmental degradation and destruction by humans, its population has decreased sharply, therefore, has been listed as one of the "National Class II Key Protected Endangered Plants" in China. T. ciliata has great economic value that its wood is often used to produce high-end furniture, high-end musical instruments and high-quality crafts [2], and more importantly, it is often used as a medicinal plant [3]. Its roots, stems, and leaves have rich chemical substances. Compounds that have been isolated from T. ciliata include ketones, steroids and coumarins, many of which have antifungal, anti-glycation or anti-tumor activity [4–7], and the flower extract has a therapeutic effect on gastric ulcers [8]. However, the yield of compounds isolated from T. ciliata is low. In addition, in previous research, it has been found that T. ciliata is very susceptible to the moth pest Hypsipyla robusta Moore [9] which eats mainly the young stems and causes the hollow branches to fail to grow and die in some cases. This pest is not only a regional issue in China, but also a worldwide problem. In some of the main areas where H. robusta is distributed, such as Australia and Brazil, T. ciliata also faces serious damage from H. robusta [10–12]. At present, there are no chemical and physical methods for effectively preventing and controlling H. robusta, and those that are available usually consume a lot of labor work and material resources, therefore, are not applicable to large-scale forest plantations [13]. Obtaining insect-resistant plants through molecular breeding may help to solve the problem of damage from the pest. In order to synthesize a desired compound related to the resistance mechanism, it is necessary to first explore the pathway and its related regulatory genes in those biological pathways [14, 15]. In exploring biosynthetic and insect-resistance mechanisms in T. ciliata, gene expression analysis is one of the most important steps. So far, the knowledge base ICG (http://icg.big.ac.cn) has collected reference genes from more than 120 plant species including Arabidopsis [16], peanut [17], cucumber [18], soybean [19], but it does not include T. ciliata. Nor are there any literature references about T. ciliata housekeeping genes which can be used for the standardization of gene expression.
RT-qPCR has advantages, including good repeatability, high sensitivity, accurate quantification, and fast reaction, making it a powerful tool for carrying out the entire PCR process and the most commonly used method for determining gene expression levels [20]. However, RT-qPCR can be affected by multiple sources of error, such as the amount of starting material, the integrity of the RNA, and the efficiency of the enzymatic reaction. It is therefore necessary to introduce a relatively stable expressed housekeeping gene as a reference for correction and standardization, so as to control the unnecessary errors generated within and between samples [21].
The commonly used housekeeping genes are those encoding actin (ACT), glyceraldehyde-3 phosphate dehydrogenase (GAPDH), and tubulin (TUB), because they are considered to be consistently expressed under all conditions [22]. However, more and more studies are now reporting that genes that are stably expressed across different tissues, different experimental conditions, and different species do not exist. In order to ensure the accuracy of an experiment, it is important to evaluate the reference genes to select those suitable reference genes for specific experimental conditions [23]. Software packages are widely used to assess the stability of expression of candidate reference genes, and determine the one or more best choices include geNorm [24], NormFinder [25], BestKeeper [26]. Many researchers have used these algorithms to successfully identify reference genes in different species [27, 28]. In species for which a reference genome sequence is available, which have generally been studied in depth, the use of reference genes in expression analysis has greatly facilitated research into plant development and evolutionary mechanisms [29].
In this study, 20 candidate genes from T. ciliata transcriptome database generated by our group were screened: actin 7 (ACT7), phosphoglycerate kinase (PGK), 60S ribosomal protein L13 (60S-13) and L18 (60S-18), histone deacetylase 1 (HIS1) and 6 (HIS6), protein phosphatase 2 C57 (PP2C57) and C59 (PP2C59), ubiquitin-conjugating enzyme E2 5B (UBC5B) and 17 (UBC17), S-adenosylmethionine decarboxylase proenzyme (SAMDC), elongation factor 1 (EF1) and 2 (EF2), peptidyl-prolyl cis-trans isomerase CYP95 (PPIA95) and CYP26-2 (PPIA26), 18S rRNA (18S), tubulin alpha-3 chain (TUB-α), tubulin beta-5 chain (TUB-β), membrane-anchored ubiquitin-fold protein 1 (MUB1), and TIP41-like protein (TIP41). The study was carried out to determine the most suitable T. ciliata candidate gene(s) as the reference(s) for gene expression analysis using RT-qPCR technique under specific conditions including different tissues (mature leaves, young leaves, flowers, shoots and young stems) and treatments (4℃, MeJA, PEG6000 and H. robusta). In addition, TcMYB3 gene was used to confirm the reliability and validity of the reference genes screened. MYB proteins, which constitute one of the largest family of transcription factors in plants, play important roles in plant growth and development, biotic and abiotic stress responses, and circadian rhythm regulation [30, 31]. For example, the R2R3 MYB transcription factor MdMYB30 modulates plant resistance against pathogens, and Arabidopsis transcription factor MYB102 increases plant susceptibility to aphids [32, 33]. Our research provided the best reference genes for use in RT-qPCR analysis of T. ciliata under different conditions, laying a foundation for studying molecular mechanisms in T. ciliata through gene expression analysis.