基因剂量效应影响三倍体桉树叶绿素代谢转录调控网络的作用机制解析

Gene dosage effect caused by polyploidization is one of the crucial reasons for the increase of chlorophyll content in triploid plants. However, it is difficult to distinguish the influence of heterozygosity effect and gene dosage effect based on the previous studies using allopolyploid. Mechanism of gene dosage effect on chlorophyll content of triploid has not been clarified yet. Therefore, given the increase of chlorophyll content of self-crossing triploid is mainly affected by gene dosage effect, this research project will use a diploid Eucalyptus urophylla and its self-crossing triploid progenies obtained from previous studies as the research materials. The characteristics of changes in chlorophyll metabolism-related gene expression and the regularity of transcriptional regulation affected by polyploidization will be analyzed using combined analysis based on the results of transcriptome sequencing, small RNA sequencing and degradation sequencing. The superior regulatory factors in the transcriptional regulatory network which are sensitive to gene dosage effect will be identified by weighted gene co-expression network analysis (WGCNA) and multilayer hierarchical gene regulatory network (ML-hGRNs) analysis. In conclusion, this research project will reveal the mechanism of gene dosage effect on the chlorophyll metabolism-related transcriptional regulatory network in triploid Eucalyptus, and provide theoretical basis for revealing the molecular mechanism of the formation of ploidy advantages in polyploid plants.

多倍化产生的基因剂量效应，是引起三倍体植物叶绿素含量显著增加的重要原因之一。然而，以往以异源三倍体为材料的研究难以区分基因剂量效应与杂合性效应的影响，基因剂量效应影响三倍体叶绿素含量的作用机制尚未被完整揭示。为此，本项目拟以前期研究获得的尾叶桉自交三倍体子代及其唯一的二倍体亲本为材料，利用自交三倍体叶绿素含量增加主要源于基因剂量效应作用的特点，采用转录组、sRNA和降解组高通量测序联合分析的方法，重点剖析由倍性水平改变引起的叶绿素合成和降解代谢相关基因表达水平及转录调控模式的变化规律；通过构建WGCNA共表达网络和多层次分层基因调控网络（ML-hGRNs），识别验证对基因剂量效应敏感的关键高层调控因子；在此基础上解析基因剂量效应影响三倍体桉树叶绿素代谢转录调控网络的作用机制，为全面揭示多倍体植物倍性优势形成的分子机制提供理论依据。

基因剂量效应是引起多倍体植物性状变异的重要原因之一，其背后的分子机制尚未被全面揭示。本项目以尾叶桉自交三倍体子代及其唯一的二倍体亲本为材料，采用转录组、sRNA和降解组高通量测序联合分析等方法，探究倍性水平不同引起的叶绿素代谢相关基因表达和调控模式变化规律，筛选基因剂量效应敏感型关键调控因子，解析基因剂量效应影响三倍体桉树叶绿素代谢相关转录调控网络的作用机制。研究发现，尾叶桉自交三倍体的叶绿素含量显著高于二倍体对照，染色体组数量的增加可以引起幼叶至成熟功能叶发育阶段参与叶绿素合成的CHLD、HEMC、HEME、CRD、POR等基因和参与叶绿素循环的CAO、HCAR、NYC1等基因的上调表达，以及衰老阶段参与叶绿素降解的PAO、SGR等基因的下调表达；利用构建的lncRNA-miRNA-mRNA多层次分层基因调控网络，进一步明确了基因剂量效应是通过引起Eucgr.K01056（GLK1）、Eucgr.C03048（CGA1）、Eucgr.A00908（FHY3）、Eucgr.J00796（bHLH130）这4个关键高层调控因子（转录因子）和CHLI、CHLD、CHLH、HEMA、POR、PIF3/PIF4等14个叶绿素代谢通路基因，以及参与调控的miRNA和lncRNA的表达改变，通过促进幼叶至成熟功能叶发育阶段叶绿素合成和抑制衰老阶段叶绿素降解促进三倍体尾叶桉叶绿素的积累；其中，miR3630和miR171m靶向的Eucgr.K01056（GLK1）被鉴定为对基因剂量效应敏感的叶绿素生物合成正调节因子，生长素介导的Eucgr.K01056（GLK1）在三倍体中的上调表达增强了Eucgr.F01098（POR）的表达，可能是基因剂量效应引起尾叶桉三倍体叶绿素含量增加的重要机制。