基于ssRNA-seq的木薯抗旱lncRNA的挖掘及相关基因调控网络的研究

Cassava is a typical drought-resistant crop. Our previous study found that transcription factors (TFs) and genes involved in ABA signaling pathways were co-expressed and participated in drought stress in cassava. It has been demonstrated that long non-coding RNA (lncRNA) plays key roles in gene expression regulation under drought condition, but the functions and related regulatory networks (e.g., whether the lncRNA participated in drought stress through ABA signaling pathways or transcription factor regulation) remain unclear in cassava. In this project, strand-specific RNA-seq (ssRNA-seq) technology is employed to explore the lncRNAs and differentially expressed genes (or their mRNAs) at the transcriptional level. Subsequently, their related gene regulatory networks are built through “lncRNA-mRNA” co-expression analysis. For the key lncRNA candidates, which are involved in TF/ABA enriched modules, will be selected for the functional validation in cassava. The findings will provide new insights into the regulatory networks of lncRNA-involved drought stress in cassava, and will also provide solid theoretical bases and candidate genes for the development of new drought-resistant cassava varieties.

木薯是典型的抗旱作物。我们前期研究发现ABA信号途径基因与转录因子（TF）共表达，共同参与木薯干旱胁迫应答。长链非编码RNA（lncRNA）在植物抗旱中扮演着重要角色。但是，lncRNA是否通过TF和ABA信号途径参与木薯抗旱调控尚不清楚。本项目拟以链特异性RNA-seq（ssRNA-seq）技术在转录组水平挖掘木薯抗旱相关的lncRNA和差异表达基因，通过基因共表达来解析相关的基因调控网络。之后重点关注TF和ABA代谢富集的模块，选取其中关键的lncRNA进行功能验证。本项目的开展将为揭示lncRNA参与木薯抗旱的基因调控网络提供新视角，同时也为木薯抗旱新品种的培育提供理论基础和候选基因。

木薯是典型的抗旱作物。为初步研究长链非编码RNA（lncRNA）在木薯抗旱中的作用，本项目通过PEG-6000模拟干旱，在0、3、24 h分别构建了木薯叶片（包括未展开叶、第一片完全展开叶和老叶）和根的ssRNA-seq文库并测序，发掘了833个lncRNA，并分析了其染色体位置、转录本长度、和外显子数目的分布特征。经统计分析，共发现124个差异表达lncRNA和5187个差异表达基因。共表达分析将这些基因/lncRNA分为11个主要模块（M1-M11），包含3618个基因和46个lncRNA，其中有8个模块（除了M1、M2、和M6）包含了至少1个lncRNA，说明lncRNA参与了木薯干旱胁迫应答。功能富集分析表明，细胞周期、细胞分化和细胞壁合成相关基因主要在未展开叶中表达，其表达量被干旱胁迫显著抑制了（M3）；光合作用相关基因主要在第一片完全展开叶和老叶中表达，其表达量也被干旱胁迫显著的抑制了（M5和M7）；激素相关基因主要在根中表达，例如ABA相关基因被干旱显著的诱导了（M10）。特别是，M10中lncRNA-TCONS_00060863与ABA转运子PDR12和ABA合成基因ABA2共表达；此外，它还和参与ABA信号调控的转录因子WRKY75共表达，表明TCONS_00060863是一个非常关键的lncRNA，它可能与WRKY75互作通过ABA信号参与木薯干旱胁迫。之后，我们用qRT-PCR方法对这些基因和lncRNA的表达进行了验证。研究结果为揭示lncRNA参与木薯抗旱的基因调控网络提供了新视角，同时也为木薯抗旱新品种的培育提供理论基础和候选基因。