毛白杨次生维管系统再生过程的基因表达

Gene Expression of Secondary Vascular System Regeneration in Populus tomentosa

【目的】次生维管系统再生能够在树干维管系统丧失后通过去分化、再分化或转分化等组织修复手段实现维管组织的再生。为了鉴定和研究次生维管系统发育相关的重要基因和调控元件,本文利用毛白杨次生维管再生系统,分析了不同再生时期的基因表达模式,为进一步揭示木材形成的基因调控机制奠定基础。【方法】将4年生毛白杨无性系在形成层活动旺盛期进行环剥取样。对剥皮后的树皮内侧和树干表面的样本,以及再生第7、10、14、18和21天5个不同时期获取的再生材料进行高通量转录组分析,并利用基因共表达分析(WGCNA)方法,得到与不同再生时期密切相关的模块,并对这些特异表达的基因进行表达网络和生物学功能分析。【结果】将第0天(剥皮当天)树皮内侧样本与树干表面样本、再生第7天样本与树干表面样本,以及相邻再生时期的样本进行表达差异分析。对得到的14202个差异基因进行WGCNA分析,构建基因共表达网络,获得与维管再生过程相关的10个共表达模块。其中,Grey60模块的基因在再生第7天样本和树皮内侧样本中特异表达,主要涉及DNA复制、有丝分裂、细胞分裂和微管运动等功能;该模块还含有大量与表观遗传调控、细胞周期相关的基因,它们在再生第7天的高表达可能激活了脱分化木质部细胞的增殖和组织类型的改变,使其获得再分化能力。此外,Pink模块的基因在第0天成熟维管组织样本中的表达量极低,但在再生过程中表达呈上升趋势,到第21天达到最高值;这些基因主要参与细胞分裂、细胞壁修饰、韧皮部发育、碳水化合物代谢、DNA的转录调控等相关的生物学途径。Pink模块中与韧皮部分化相关的标记基因,比如APL、NAC45/86、DOF、BSPA、PP2和SUS的表达被重新激活,与调节形成层细胞增殖和分化的CLE41/44和CLV1一样,表达量都是从再生第10天开始逐渐增加。到次生维管系统再生后期的第18天和第21天,形成层已经完成了结构重构并能进行细胞的分裂、分化,此时木质部标记基因和次生壁调控因子有较高的表达。【结论】毛白杨次生维管系统再生早期,脱分化的木质部细胞通过表观遗传和细胞周期调控重新获得细胞分生能力;之后,韧皮部的细胞分化程序被启动,再生形成层开始细胞增殖和分化;到再生后期,通过相关基因调控,形成层再分化木质部和韧皮部。通过对次生维管组织再生过程中基因的表达分析和调控模块的鉴定,进一步验证了次生维管再生的遗传调控基础,也可为揭示植物维管组织再生的调控机制奠定基础。

【Objective】The secondary vascular system(SVS)regeneration can regenerate vascular tissue through tissue repairing such as past differentiation,re-differentiation or transdifferentiation after the loss of vascular system.In order to identify and study the important genes and regulatory elements related to the development of SVS,we analyzed the gene expression patterns at different regeneration stages of SVS regeneration of Populus tomentosa.It laid a foundation for further revealing the gene regulation mechanism of wood formation.【Method】Four-year-old P.tomentosa clones were girdled and sampled during the active period of cambium.High throughput transcriptome analysis was performed on the inner bark and trunk surface samples after girdling,as well as the regenerated tissue obtained on the 7th,10th,14th,18th and 21st days after the girdling.Weighted gene co-expression network analysis(WGCNA)was used to obtain the modules closely related to different regeneration stages,and the expression network and biological function of these specifically expressed genes were analyzed.【Result】The 14202 differentially expressed genes,obtained by comparing differences between the samples of inner bark and trunk surface,7th day and trunk surface,and adjacent regeneration periods,were analyzed by WGCNA.The gene co-expression network was constructed and 10 modules related to the regeneration of SVS were obtained.Among them,the genes in Grey60 module,which were specifically expressed in the samples on the 7th day of regeneration and inner bark,mainly involved in DNA replication,mitosis,cell division and microtubule movement.The module also contained a large number of genes related to epigenetics and cell cycle.Their high expression on the 7th day of regeneration may activate the proliferation of dedifferentiated xylem cells and the change of tissue type,which enabled them to re-differentiate.In addition,the expression values of genes in the Pink module were very low in the mature vascular tissue samples on day 0,but they showed an upward trend in the process of regeneration,and reached the highest values on 21th day.These genes were mainly involved in cell division,cell wall modification,phloem development,carbohydrate metabolism,DNA transcription regulation and other related biological pathways.In the Pink module,the expression of some phloem related marker genes,such as APL,NAC45/86,DOF,BSPA,PP2 and SUS,were reactivated.Same as CLE41/44 and CLV1 that can regulate the proliferation and differentiation of cambium cells,their expression level increased gradually from the 10th day.At the late stage of SVS regeneration on the 18th and 21st day,the re-differentiated cambium had completed structural reconstruction and could undergo cell division and differentiation.The xylem marker genes and secondary wall regulatory factors were highly expressed at this stage.【Conclusion】At the early stage of SVS regeneration of P.tomentosa,the dedifferentiated xylem cells regained the ability of cell division through epigenetic and cell cycle regulation;then the cell differentiation of phloem was started,and the regeneration cambium began cell proliferation and differentiation;at the late stage of regeneration,the cambium re-differentiated into xylem and phloem through the regulation of related genes.Through the analysis of gene expression and identification of regulatory modules in the process of SVS regeneration,we further verified the genetic regulatory basis of SVS regeneration,and also laid a foundation for revealing the regulatory mechanism of plant vascular tissue regeneration.