Yangdong Wang

Selection of Reliable Reference Genes for Gene Expression Studies Using Real-Time PCR in Tung Tree during Seed Development

Quantitative real-time PCR (RT-qPCR) has become an accurate and widely used technique to analyze expression levels of selected genes. It is very necessary to select appropriate reference genes for gene expression normalization. In the present study, we assessed the expression stability of 11 reference genes including eight traditional housekeeping genes and three novel genes in different tissues/organs and developing seeds from four cultivars of tung tree. All 11 reference genes showed a wide range of Ct values in all samples, indicating that they differently expressed. Three softwares – geNorm, NormFinder and BestKeeper – were used to determine the stability of these references except for ALB (2S albumin), which presented a little divergence. The results from the three softwares showed that ACT7 (Actin7a), UBQ (Ubiquitin), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and EF1α (elongation factor 1-α) were the most stable reference genes across all of the tested tung samples and tung developing seeds, while ALB (2S albumin) was unsuitable as internal controls. ACT7, EF1β (elongation factor1-beta), GAPDH and TEF1 (transcription elongation factor 1) were the top four choices for different tissues/organs whereas LCR69 did not favor normalization of RT-qPCR in these tissues/organs. Meanwhile, the expression profiles of FAD2 and FADX were realized using stable reference genes. The relative quantification of the FAD2 and FADX genes varied according to the internal controls and the number of internal controls. The results further proved the importance of the choice of reference genes in the tung tree. These stable reference genes will be employed in normalization and quantification of transcript levels in future expression studies of tung genes.

Transcriptome Sequencing and Expression Analysis of Terpenoid Biosynthesis Genes in Litsea cubeba

Background Aromatic essential oils extracted from fresh fruits of Litsea cubeba (Lour.) Pers., have diverse medical and economic values. The dominant components in these essential oils are monoterpenes and sesquiterpenes. Understanding the molecular mechanisms of terpenoid biosynthesis is essential for improving the yield and quality of terpenes. However, the 40 available L. cubeba nucleotide sequences in the public databases are insufficient for studying the molecular mechanisms. Thus, high-throughput transcriptome sequencing of L. cubeba is necessary to generate large quantities of transcript sequences for the purpose of gene discovery, especially terpenoid biosynthesis related genes. Results Using Illumina paired-end sequencing, approximately 23.5 million high-quality reads were generated. De novo assembly yielded 68,648 unigenes with an average length of 834 bp. A total of 38,439 (56%) unigenes were annotated for their functions, and 35,732 and 25,806 unigenes could be aligned to the GO and COG database, respectively. By searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG), 16,130 unigenes were assigned to 297 KEGG pathways, and 61 unigenes, which contained the mevalonate and 2-C-methyl-D-erythritol 4-phosphate pathways, could be related to terpenoid backbone biosynthesis. Of the 12,963 unigenes, 285 were annotated to the terpenoid pathways using the PlantCyc database. Additionally, 14 terpene synthase genes were identified from the transcriptome. The expression patterns of the 16 genes related to terpenoid biosynthesis were analyzed by RT-qPCR to explore their putative functions. Conclusion RNA sequencing was effective in identifying a large quantity of sequence information. To our knowledge, this study is the first exploration of the L. cubeba transcriptome, and the substantial amount of transcripts obtained will accelerate the understanding of the molecular mechanisms of essential oils biosynthesis. The results may help improve future genetic and genomics studies on the molecular mechanisms behind the chemical composition of essential oils in L. cubeba fruits.

Chemical Composition of Essential Oils of Litsea cubeba Harvested from Its Distribution Areas in China

Litsea cubeba (Lour.) Pers. is a promising industrial crop with fruits rich in essential oils. The chemical composition of essential oils of L. cubeba (EOLC) were determined for fruits harvested from eight regions in China. The overall essential oil content, obtained by hydrodistillation and analyzed by gas chromatography–mass spectrometry (GC-MS), ranged from 3.04% to 4.56%. In total, 59 compounds were identified, the dominant components being monoterpenes (94.4–98.4%), represented mainly by neral and geranial (78.7–87.4%). D-limonene was unexpectedly a lesser constituent (0.7–5.3%) in fruits, which differed from previous reports (6.0–14.6%). Several components were only detected in certain regions and compounds such as o-cymene and eremophilene have never before been reported in EOLC. These results demonstrate significant regional variation in the chemical composition of EOLC. This investigation provides important information with regard to the bioactivity, breeding work and industrial applications of L. cubeba.

Identification of appropriate reference genes for normalizing transcript expression by quantitative real-time PCR in Litsea cubeba

Quantitative real-time PCR has emerged as a highly sensitive and widely used method for detection of gene expression profiles, via which accurate detection depends on reliable normalization. Since no single control is appropriate for all experimental treatments, it is generally advocated to select suitable internal controls prior to use for normalization. This study reported the evaluation of the expression stability of twelve potential reference genes in different tissue/organs and six fruit developmental stages of Litseacubeba in order to screen the superior internal reference genes for data normalization. Two softwares—geNorm, and NormFinder—were used to identify stability of these candidate genes. The cycle threshold difference and coefficient of variance were also calculated to evaluate the expression stability of candidate genes. F-BOX, EF1α, UBC, and TUA were selected as the most stable reference genes across 11 sample pools. F-BOX, EF1α, and EIF4α exhibited the highest expression stability in different tissue/organs and different fruit developmental stages. Besides, a combination of two stable reference genes would be sufficient for gene expression normalization in different fruit developmental stages. In addition, the relative expression profiles of DXS and DXR were evaluated by EF1α, UBC, and SAMDC. The results further validated the reliability of stable reference genes and also highlighted the importance of selecting suitable internal controls for L. cubeba. These reference genes will be of great importance for transcript normalization in future gene expression studies on L. cubeba.

Biology and chemistry of Litsea cubeba, a promising industrial tree in China

Litsea cubeba (Lour.) Pers. (Lauraceae) is a promising industrial crop. The plant is rich in valuable essential oil, which is widely used as an ingredient in ionone flavors, botanical insecticides, food spices and personal-care products, and the plant extract has been used in traditional Chinese medicine. Furthermore, the potential for producing biodiesel through transesterification of L. cubeba kernel oil holds great promise as a low-cost bioenergy. Based on the investigation of L. cubeba genetic resources in the main distribution in China, the article presents an overview of current advances and future perspectives in the study and use of L. cubeba, including basic biology, chemistry and various industrial uses. We intend to provide data and guidance for future large-scale cultivation and creation of deep processing techniques to meet the growing commercial demands for L. cubeba.

FAD2-DGAT2 genes coexpressed in endophytic Aspergillus fumigatus derived from tung oilseeds.

Recent efforts to genetically engineer plants that contain fatty acid desaturases to produce valuable fatty acids have made only modest progress. Diacylglycerol acyltransferase 2 (DGAT2), which catalyzes the final step in triacylglycerol (TAG) assembly, might potentially regulate the biosynthesis of desired fatty acids in TAGs. To study the effects of tung tree (Vernicia fordii) vfDGAT2 in channeling the desired fatty acids into TAG, vfDGAT2 combined with the tung tree fatty acid desaturase-2 (vfFAD2) gene was co-introduced into Aspergillus fumigatus, an endophytic fungus isolated from healthy tung oilseed. Two transformants coexpressing vfFAD2 and vfDGAT2 showed a more than 6-fold increase in linoleic acid production compared to the original A. fumigatus strain, while a nearly 2-fold increase was found in the transformant expressing only vfFAD2. Our data suggest that vfDGAT2 plays a pivotal role in promoting linoleic acid accumulation in TAGs. This holds great promise for further genetic engineering aimed at producing valuable fatty acids.

Divergent Expression Patterns in Two Vernicia Species Revealed the Potential Role of the Hub Gene VmAP2/ERF036 in Resistance to Fusarium oxysporum in Vernicia montana

Tung oil tree (Vernicia fordii) is a promising industrial oil crop; however, this tree is highly susceptible to Fusarium wilt disease. Conversely, Vernicia montana is resistant to the pathogen. The APETALA2/ethylene-responsive element binding factor (AP2/ERF) transcription factor superfamily has been reported to play a significant role in resistance to Fusarium oxysporum. In this study, comprehensive analysis identified 75 and 81 putative Vf/VmAP2/ERF transcription factor-encoding genes in V. fordii and V. montana, respectively, which were divided into AP2, ERF, related to ABI3 and VP1 (RAV) and Soloist families. After F. oxysporum infection, a majority of AP2/ERF superfamily genes showed strong patterns of repression in both V. fordii and V. montana. We then identified 53 pairs of one-to-one orthologs in V. fordii and V. montana, with most pairs of orthologous genes exhibiting similar expression in response to the pathogen. Further investigation of Vf/VmAP2/ERF gene expression in plant tissues indicated that the pairs of genes with different expression patterns in response to F. oxysporum tended to exhibit different tissue profiles in the two species. In addition, VmAP2/ERF036, showing the strongest interactions with 666 genes, was identified as a core hub gene mediating resistance. Moreover, qRT-PCR results indicated VmAP2/ERF036 showed repressed expression while its orthologous gene VfAP2/ERF036 had the opposite expression pattern during pathogen infection. Overall, comparative analysis of the Vf/VmAP2/ERF superfamily and indication of a potential hub resistance gene in resistant and susceptible Vernicia species provides valuable information for understanding the molecular basis and selection of essential functional genes for V. fordii genetic engineering to control Fusarium wilt disease.

Identification and characterization of NF-YB family genes in tung tree

The NF-YB transcription factor gene family encodes a subunit of the CCAAT box-binding factor (CBF), a highly conserved trimeric activator that strongly binds to the CCAAT box promoter element. Studies on model plants have shown that NF-YB proteins participate in important developmental and physiological processes, but little is known about NF-YB proteins in trees. Here, we identified seven NF-YB transcription factor-encoding genes in Vernicia fordii, an important oilseed tree in China. A phylogenetic analysis separated the genes into two groups; non-LEC1 type (VfNF-YB1, 5, 7, 9, 11, 13) and LEC1-type (VfNF-YB 14). A gene structure analysis showed that VfNF-YB 5 has three introns and the other genes have no introns. The seven VfNF-YB sequences contain highly conserved domains, a disordered region at the N terminus, and two long helix structures at the C terminus. Phylogenetic analyses showed that VfNF-YB family genes are highly homologous to GmNF-YB genes, and many of them are closely related to functionally characterized NF-YBs. In expression analyses of various tissues (root, stem, leaf, and kernel) and the root during pathogen infection, VfNF-YB1, 5, and 11 were dominantly expressed in kernels, and VfNF-YB7 and 9 were expressed only in the root. Different VfNF-YB family genes showed different responses to pathogen infection, suggesting that they play different roles in the pathogen response. Together, these findings represent the first extensive evaluation of the NF-YB family in tung tree and provide a foundation for dissecting the functions of VfNF-YB genes in seed development, stress adaption, fatty acid synthesis, and pathogen response.

Genome-Wide Identification and Characterization of the LRR-RLK Gene Family in Two Vernicia Species

Leucine-rich repeat receptor-like kinases (LRR-RLKs) make up the largest group of RLKs in plants and play important roles in many key biological processes such as pathogen response and signal transduction. To date, most studies on LRR-RLKs have been conducted on model plants. Here, we identified 236 and 230 LRR-RLKs in two industrial oil-producing trees: Vernicia fordii and Vernicia montana, respectively. Sequence alignment analyses showed that the homology of the RLK domain (23.81%) was greater than that of the LRR domain (9.51%) among the Vf/VmLRR-RLKs. The conserved motif of the LRR domain in Vf/VmLRR-RLKs matched well the known plant LRR consensus sequence but differed at the third last amino acid (W or L). Phylogenetic analysis revealed that Vf/VmLRR-RLKs were grouped into 16 subclades. We characterized the expression profiles of Vf/VmLRR-RLKs in various tissue types including root, leaf, petal, and kernel. Further investigation revealed that Vf/VmLRR-RLK orthologous genes mainly showed similar expression patterns in response to tree wilt disease, except 4 pairs of Vf/VmLRR-RLKs that showed opposite expression trends. These results represent an extensive evaluation of LRR-RLKs in two industrial oil trees and will be useful for further functional studies on these proteins.

Comparative Transcriptomics Atlases Reveals Different Gene Expression Pattern Related to Fusarium Wilt Disease Resistance and Susceptibility in Two Vernicia Species

Vernicia fordii (tung oil tree) is a promising industrial crop. Unfortunately, the devastating Fusarium wilt disease has caused its great losses, while its sister species (Vernicia montana) is remarkably resistant to this pathogen. However, the genetic mechanisms underlying this difference remain largely unknown. We here generated comparative transcriptomic atlases for different stages of Fusarium oxysporum infected Vernicia root. The transcriptomes of V. fordii and V. montana were assembled de novo and contained 258,430 and 245,240 non-redundant transcripts with N50 values of 1776 and 2452, respectively. A total of 44,310 pairs of putative one-to-one orthologous genes were identified in Vernicia species. Overall, the vast majority of orthologous genes shared a remarkably similar expression mode. The expression patterns of a small set of genes were further validated by quantitative real-time PCR. Moreover, 157 unigenes whose expression significantly correlated between the two species were defined, and gene set enrichment analysis indicated roles in increased defense response and in jasmonic and salicylic acid signaling responses during pathogen attack. Co-expression network analysis further identified the 17 hub unigenes, such as the serine/threonine protein kinase D6PK, leucine-rich repeat receptor-like kinase (LRR-RLK), and EREBP transcription factor, which play essential roles in plant pathogen resistance. Intriguingly, the expression of most hub genes differed significantly between V. montana and V. fordii. Based on our results, we propose a model to describe the major molecular reactions that underlie the defense responses of resistant V. montana to F. oxysporum. These data represent a crucial step toward breeding more pathogen-resistant V. fordii.