Genome-Wide Identification of Differentially Expressed Genes Associated with the High Yielding of Oleoresin in Secondary Xylem of Masson Pine (<i>Pinus massoniana</i> Lamb) by Transcriptomic Analysis

Masson pine is an important timber and resource for oleoresin in South China. Increasing yield of oleoresin in stems can raise economic benefits and enhance the resistance to bark beetles. However, the genetic mechanisms for regulating the yield of oleoresin were still unknown. Here, high-throughput sequencing technology was used to investigate the transcriptome and compare the gene expression profiles of high and low oleoresin-yielding genotypes. A total of 40,690,540 reads were obtained and assembled into 137,499 transcripts from the secondary xylem tissues. We identified 84,842 candidate unigenes based on sequence annotation using various databases and 96 unigenes were candidates for terpenoid backbone biosynthesis in pine. By comparing the expression profiles of high and low oleoresin-yielding genotypes, 649 differentially expressed genes (DEGs) were identified. GO enrichment analysis of DEGs revealed that multiple pathways were related to high yield of oleoresin. Nine candidate genes were validated by QPCR analysis. Among them, the candidate genes encoding geranylgeranyl diphosphate synthase (GGPS) and (-)-alpha/beta-pinene synthase were up-regulated in the high oleoresin-yielding genotype, while tricyclene synthase revealed lower expression level, which was in good agreement with the GC/MS result. In addition, DEG encoding ABC transporters, pathogenesis-related proteins (PR5 and PR9), phosphomethylpyrimidine synthase, non-specific lipid-transfer protein-like protein and ethylene responsive transcription factors (ERFs) were also confirmed to be critical for the biosynthesis of oleoresin. The next-generation sequencing strategy used in this study has proven to be a powerful means for analyzing transcriptome variation related to the yield of oleoresin in masson pine. The candidate genes encoding GGPS, (-)-alpha/beta-pinene, tricyclene synthase, ABC transporters, non-specific lipid-transfer protein-like protein, phosphomethylpyrimidine synthase, ERFs and pathogen responses may play important roles in regulating the yield of oleoresin. These DEGs are worthy of special attention in future studies.马尾松（Masson pine）是中国南方重要的用材及松脂资源树种。提升树干的松脂产量，既可提高经济效益，又能增强其对小蠹虫的抗性。然而，当前调控松脂产量的遗传机制仍未明确。本研究采用高通量测序技术，对不同松脂产量基因型的转录组（transcriptome）进行探究，并比较二者的基因表达谱。从次生木质部组织中，共获取40,690,540条测序读段（reads），经组装得到137,499条转录本。基于多数据库序列注释，我们共鉴定出84,842个候选单基因（unigene），其中96个候选单基因参与松树的萜类骨架生物合成。通过比较高低松脂产量基因型的表达谱，共鉴定出649个差异表达基因（differentially expressed genes, DEGs）。对DEGs进行GO富集分析（Gene Ontology enrichment analysis）显示，多条通路与松脂高产相关。通过实时荧光定量PCR（QPCR）验证了9个候选基因，其中编码牻牛儿基牻牛儿基二磷酸合酶（geranylgeranyl diphosphate synthase, GGPS）与(-)-α/β-蒎烯合酶的候选基因在高松脂产量基因型中呈上调表达，而三环烯合酶的表达水平较低，这一结果与气相色谱-质谱联用（GC/MS）分析结果一致。此外，编码ABC转运蛋白（ABC transporters）、病程相关蛋白（pathogenesis-related proteins, PR5与PR9）、磷酸甲基嘧啶合酶、非特异性脂质转移蛋白样蛋白以及乙烯响应转录因子（ethylene responsive transcription factors, ERFs）的DEGs也被证实对松脂生物合成至关重要。本研究采用的二代测序策略，已被证明是分析马尾松松脂产量相关转录组变异的有效手段。编码GGPS、(-)-α/β-蒎烯合酶、三环烯合酶、ABC转运蛋白、非特异性脂质转移蛋白样蛋白、磷酸甲基嘧啶合酶、ERFs以及参与病原响应的基因，可能在调控松脂产量中发挥重要作用。这些DEGs值得在未来的研究中重点关注。