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Identification, Expression, and Phylogenetic Analyses of Terpenoid Biosynthesis-Related Genes in Secondary Xylem of Loblolly Pine ( Pinus taeda L.) Based on Transcriptome Analyses

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Identification, Expression, and Phylogenetic Analyses of Terpenoid Biosynthesis-Related Genes in Secondary Xylem of Loblolly Pine ( Pinus taeda L.) Based on Transcriptome Analyses

Jipeng Mao et al. PeerJ.

Abstract

Loblolly pine (Pinus taeda L.) is one of the most important species for oleoresin (a mixture of terpenoids) in South China. The high oleoresin content of loblolly pine is associated with resistance to bark beetles and other economic benefits. In this study, we conducted transcriptome analyses of loblolly pine secondary xylem to gain insight into the genes involved in terpenoid biosynthesis. A total of 372 unigenes were identified as being critical for oleoresin production, including genes for ATP-binding cassette (ABC) transporters, the cytochrome P450 (CYP) protein family, and terpenoid backbone biosynthesis enzymes. Six key genes involved in terpenoid biosynthetic pathways were selected for multiple sequence alignment, conserved motif prediction, and phylogenetic and expression profile analyses. The protein sequences of all six genes exhibited a higher degree of sequence conservation, and upstream genes were relatively more conserved than downstream genes in terpenoid biosynthetic pathways. The N-terminal regions of these sequences were less conserved than the C-terminal ends, as the N-terminals were quite diverse in both length and composition. The phylogenetic analyses revealed that most genes originated from gene duplication after species divergence, and partial genes exhibited incomplete lineage sorting. In addition, the expression profile analyses showed that all six genes exhibited high expression levels during the high-oleoresin-yielding phase.

Keywords: Conserved motifs; Loblolly pine (Pinus taeda L.); Phylogenetic analysis; Terpenoid biosynthesis; Transcriptome analysis.

Conflict of interest statement

Jiehu Chen is employed by Science Corporation of Gene

Figures

Figure 1
Figure 1. Oleoresin and RNA samples collection methods.
(A) Artificial collection of oleoresin in South China. (B) Sampling site and method of secondary xylem tissue.
Figure 2
Figure 2. Length distribution.
The length distribution of 74,402 assembled unigenes in secondary xylem of Pinus taeda.
Figure 3
Figure 3. Venn diagram.
The venn diagram of 31,586 unigenes from five public database.
Figure 4
Figure 4. Assembled unigenes were functionally classificated by Gene Ontology categorization.
The unigenes corresponded to three main categories: biological process, cellular component, and molecular function.
Figure 5
Figure 5. KOG functional classification of all unigenes.
A total of 18,786 unigenes showed significant similarity to the sequences in KOG databases and were clustered into 25 categories.
Figure 6
Figure 6. Assembled unigenes were functionally classificated by KEGG classification.
The unigenes corresponded to five main categories: Cellular processes, Environmental information Processing, Genetic information Processing, Metabolism and Organismal Systems.
Figure 7
Figure 7. Distribution of conserved motifs of DXR protein sequences in 15 plant species.
Phylogenetic relationship were displayed on left, and blue, red and green lines represented gymnosperm, monocotyledon and dicotyledon, respectively. Each motif is represented by a colored box and a number, and the same number with differ letters represented the identical motif located in different sites.
Figure 8
Figure 8. Biosynthetic pathway of terpenoids (adapted from Zulak & Bohlmanu (2010), Liu et al. (2015)).
The number of unigenes homologous to gene families encoding these enzymes was provided in parentheses.
Figure 9
Figure 9. Bayesian phylogenetic analysis of the DXR, DXS, HMGR, IPPI, GGPS and FPS genes in 15 plant species.
They bootstrap values were given in present and the scale bar indicate 0.05, 0.05, 0.1, 0.2, 0.2 and 0.05 substitutions per site, respectively. (A) phylogenetic tree of DXR gene with JTT+I+G model, (B) phylogenetic tree of DXS gene with JTT+I+G model, (C) phylogenetic tree of GGPS gene with LG+G+F model, (D) phylogenetic tree of FPS gene with LG+I+G model, (E) phylogenetic tree of HMGR gene with JTT+G+F model, (F) phylogenetic tree of IPPI gene with JTT+I+G model.
Figure 10
Figure 10. Quantitative RT-PCR analysis of six candidate genes in three different oleoresin-yielding stages (Apr., Aug. and Oct.).
(A) DXS; (B) DXR; (C) HMRG; (D) IPPI; (E) FPS; (F) GGPS. Relative expression levels of qRT-PCR calculated using Actin as the internal control were shown in the y-axis. Error bars represent SD of the mean for three biological replicates.

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Grant support

This work was supported by the National Key R&D Program of China (2017YFD0600502-3) and “948” program of State Forestry Administration P.R. China (2014-4-72). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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