A Betula luminifera transcriptome and analysis of shoot and root specific phosphate deficiency induced transcriptional and post transcriptional responses (RNA-Seq). A Betula luminifera transcriptome and analysis of shoot and root specific phosphate deficiency induced transcriptional and post transcriptional responses (RNA-Seq)

microRNAs (miRNAs) play important roles in responses to abiotic stresses, including nutrition stress, by regulating target gene expression. Phosphate (Pi) is often lacking in natural and agro-climatic environments, and plants have developed strategies to cope with low Pi (LP) availability. However, the miRNA-mediated regulation of these adaptive responses and their underlying coordinating signals are still poorly understood in forestry trees such as Betula luminifera. Four small RNA (sRNA) libraries, a mixed degradome cDNA library, and four transcriptomic libraries of B. luminifera roots and shoots treated under LP and normal conditions (CK) were constructed and sequenced using next-generation deep sequencing. sRNA sequencing analyses indicated that 66 and 60 miRNAs were differentially expressed in roots and shoots, respectively, under LP conditions. A total of 109 and 112 miRNA–target pairs were further validated in the roots and shoots, respectively, using degradome sequencing, including several novel target genes that were cleaved by isomiRNAs with lengths of 18 or 19 nucleotides, which were only differentially expressed in roots. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of differential miRNA targets indicated that the “ascorbate and aldarate metabolism” pathway responded to LP, and “circadian rhythm – plant” was specifically enriched in shoots. A comprehensive B. luminifera transcriptome derived from its roots and shoots was constructed, and a total of 76,899 unigenes were generated. A comparison of transcriptome identified 8,095 and 5,584 differentially expressed genes in roots and shoots, respectively, under LP conditions. Integrated analysis uncovered 14 and 16 miRNA–target pairs that showed negatively correlated expression profiles in roots and shoots, respectively. Moreover, a putative model of miRNA–target interaction involved in plant responses to LP stress is proposed. These results suggest that comprehensive analyses of sRNAs, degradome, and transcriptome provide a useful platform for investigating LP stress in B. luminifera, and may provide new insights into the genetic engineering of high use efficiency of Pi in forestry trees. Overall design: Four transcriptomic libraries of B. luminifera roots and shoots treated under LP and normal conditions (CK) were constructed and sequenced using next-generation deep sequencing.

微小核糖核酸（microRNAs, miRNAs）在包括营养胁迫在内的非生物胁迫响应中发挥关键作用，可通过调控靶基因表达参与上述胁迫响应过程。磷酸盐（Phosphate, Pi）在自然及农业气候环境中往往匮乏，植物已演化出多种策略以应对低磷（low Pi, LP）有效性不足的问题。然而，在光皮桦（Betula luminifera）这类林木中，miRNA介导的上述适应性响应调控机制及其潜在的协同信号通路仍不甚明晰。
本研究构建了光皮桦经LP处理及正常对照（CK）处理的根、茎组织的4个小RNA（small RNA, sRNA）文库、1个混合降解组（degradome）cDNA文库，以及4个转录组文库，并通过下一代深度测序完成测序。小RNA测序分析显示，在LP处理条件下，根组织中共有66个miRNA呈差异表达，茎组织中则有60个miRNA呈差异表达。通过降解组测序，本研究分别在根、茎组织中验证了109对和112对miRNA-靶基因互作对，其中包含若干仅在根组织中呈差异表达的18或19核苷酸长度的miRNA变体（isomiRNAs）所切割的新型靶基因。
针对差异表达miRNA靶基因的京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路富集分析显示，"抗坏血酸和乙醛酸代谢（ascorbate and aldarate metabolism）"通路响应LP胁迫，而"植物昼夜节律（circadian rhythm – plant）"通路则仅在茎组织中显著富集。本研究基于光皮桦根、茎组织构建了全面的转录组文库，共得到76899个单基因簇（unigenes）。转录组比较分析显示，在LP处理条件下，根组织中共有8095个差异表达基因，茎组织中则有5584个差异表达基因。整合分析分别在根、茎组织中发现了14对和16对呈现表达负相关模式的miRNA-靶基因互作对。此外，本研究提出了一个参与植物LP胁迫响应的miRNA-靶基因互作调控推定模型。
上述研究结果表明，针对sRNA、降解组及转录组的整合分析为光皮桦LP胁迫研究提供了一个有效的研究平台，同时也为林木高Pi利用效率的基因工程改良提供了新的研究思路。
实验设计概述：本研究通过下一代深度测序，完成了光皮桦经LP处理及正常对照（CK）处理的根、茎组织的4个转录组文库的构建与测序。