【作者】 刘志祥；

【导师】 谭晓风；

【作者基本信息】 中南林业科技大学 ， 林木遗传育种， 2013， 博士

【摘要】 杨树是重要的速生用材树种。随着毛果杨基因组测序的完成和相关遗传学和生物信息学研究的开展,目前杨树已成为公认的木本模式植物。对于许多生物学问题,例如木材形成、细胞壁次生生长、多年生生长、开花调控、生物进化、逆境胁迫响应等,杨树具有重要的研究价值。miRNA是一类广泛存在于动植物的内源调控小分子RNA,在植物中主要通过序列互补介导靶mRNA的降解以抑制靶基因的表达,从而参与植物发育调控与逆境胁迫响应。研究miRNA有助于揭示很多生命现象的分子机理,并可能应用于作物和林木的遗传改良。本文研究了杨树7个保守程度不同的miRNA基因家族的分子进化模式,进一步通过降解组分析鉴定miRNA靶基因并进行基因本体富集分析,并对2个miRNA靶基因进行了分离克隆与功能分析。主要研究结果如下：1.杨树MIR169基因家族的分子进化分析。串联重复和16.6～83.1百万年前的染色体大片段重复在毛果杨MIR169基因家族扩张中均发挥了重要作用；部分重复基因在进化过程中丢失；植物MIR169基因在单子叶植物与双子叶植物分化前已经分化为MIR169₁和MIR169₂两个家族,在杨树中又分别从MIR169₁和MIR169₂家族中分化出MIR169₄和MIR169₃家族。MIR169基因家族在表达方式上已经出现了较大的分化,其中MIR169u在多种组织中具有最高的表达活性；杨树MIR169基因家族的预测靶基因包括转录因子、蛋白激酶等多种基因,其中转录因子NF-YA是杨树MIR169主要的靶基因；MIR169基因家族可能在杨树中形成了复杂的调控网络,对杨树的生长发育和适应性等具有重要的调控作用。2.杨树MIR171基因家族的分子进化分析。杨树MIR171基因家族主要通过48～54百万年前的染色体大片段重复进行扩张,其表达方式已经出现分化,其中MIR171l/m/n在多种组织中具有最高的表达水平。MIR171基因家族可能主要通过调控GRAS转录因子和信号转导蛋白参与杨树生长发育、光信号转导和光形态建成的调控。3.杨树MR156基因家族的分子进化研究。杨树MIR156基因家族主要通过染色体大片段重复实现扩张,而串联重复对此没有贡献；不同家族成员表达方式已经分化,其中MIR156g/h/i/j表达活性最高；家族成员间启动子顺式作用元件存在差异；杨树MIR156的靶基因包括29个SBP结构域蛋白质基因,由于成熟序列存在差异,家族成员调控的靶基因也表现出差异,说明杨树MIR156基因家族成员的功能已经分化,在杨树中可能形成了复杂的调控网络。4.杨树4个新起源miRNA家族的进化。杨树MIR1444家族主要通过染色体大片段重复实现扩张,MIR1446家族主要通过染色体大片段重复和串联重复实现扩张,而非保守的MIR6425、MIR6462家族主要通过串联重复进行扩张；这4个基因家族成员的表达方式均已经分化,其中MIR1444、MIR1446在多种组织中具有较高的表达活性,而MIR6425、MIR6462仅具有较低的表达活性；MIR1446、 MIR6425、MIR6462家族内不同成员的成熟序列基本一致,其预测的靶基因也一致,而MIR1444家族中不同成员的成熟序列则出现了一定程度的分化,其预测的靶基因也不一致。对以上7个不同保守程度的miRNA基因家族的比较分析表明：起源较早、保守程度较高的miRNA基因家族成员数量较多,表达活性较高,功能分化程度较高,主要通过调控转录因子在杨树生命活动中发挥重要的调节作用；而起源较晚、保守程度较低的miRNA基因家族成员数量较少,表达活性较低,功能分化程度较低,其调控的靶基因多为酶等功能蛋白。5.杨树降解组分析与miRNA靶基因富集分析。构建了胡杨叶片降解组文库并进行高通量测序,获得42659177条原始序列,去除接头、污染序列及低质量序列后得到了34657804条干净序列用于降解组分析,共鉴定了23个miRNA靶基因。基因富集分析结果表明,胡杨叶片降解组分析中鉴定的miRNA靶基因富集于氧化还原酶活性和抗氧化活性,参与细胞氧化还原平衡的维持。这说明miRNA可能在杨树盐胁迫条件下对清除ROS、维持氧化还原的平衡具有重要的调控作用。对杨树、拟南芥和水稻高度保守和低度保守miRNA的预测靶基因分别进行单一富集分析和交叉比对。发现这三种植物的高度保守miRNA的预测靶基因都显著富集于转录因子,同时还发现杨树高度保守miRNA的预测靶基因显著富集于氧化还原过程,而低度保守miRNA靶基因富集程度较低。6.杨树miRNA靶基因的克隆与功能分析。从胡杨中克隆了MIR171的靶基因Pescll的全长cDNA, Pescll属于GRAS转录因子家族。RLM5’RACE实验证明Pescll存在miR171a-3p/b的靶位点。在盐胁迫条件下,Pescll表达水平上调,说明Pescll参与了杨树盐胁迫响应。从胡杨中克隆了MIR1444的预测靶基因Pecngcl的全长cDNA。 Pecngcl属于环核苷酸门离子通道基因家族。PeCNGCl定位于细胞膜,转Pecngcl能提高拟南芥的耐盐性。Pecngcl在叶片中表达,且其表达受盐胁迫条件的诱导,说明盐胁迫条件下Pecngcl可能对维持杨树叶片中Na+、K+的平衡发挥作用。更多还原

【Abstract】 Pupulus plants are fast-growing commercial trees. Owing to the availability of the Populus trichocarpa genome sequence and plenty of Populus-specific genetic databases, Populus is known as a model for research of woody plants. Populus serves as a reference for many fundamental scientific investigations of wood formation, secondary cell wall formation, perennial and seasonal growth, flowering, evolution and resistance physiology. MicroRNAs （miRNAs） are a class of short non-coding RNAs found widely in eukaryotic organisms. MiRNAs function as regulators of development and stress responses in plants. Study of miRNA will contribute to reveal molecular mechanisms of many phenomenon of life, and may be applied to genetic improvement of crops and forest trees. To understand the evolutionary mechanisms of miRNAs in poplar, the evolution and functional diversity of3more conserved and4less conserved poplar miRNA gene families was comparatively studied in this dissertation, and the poplar miRNA targets were investigated by degradome sequencing and gene ontology enrichment analysis, and then two miRNA target genes were cloned and their functions were analyzed. The main research contents and results are as fellows:1. Molecular evolution of the poplar MIR169gene family. Tandem duplications and chromosome segmental duplications, which happened16.6-83.1million years ago, contributed equally to the expanding of ptc-MIR169gene family, Several members of this family has disappeared during evolution. Before the differentiation of monocots and dicots, the plant MIR169family had been differentiated into two families, MIR169₁and MIR169₂, then MIR169₄and MIR169₃family differentiated from MIR169₁and MIR169₂respectively in poplar. The expression patterns diversified obviously among the gene family. MIR169u has highest expression activity in many tissues. The predicted target genes of poplar MIR169include transcription factors, protein kinases, etc. The NF-YAs are major targets of poplar MIR169. These findings suggest that the ptc-MIR169gene family is involved in complex regulatory networks, and plays significant roles in development and stress response in poplar.2. Evolutionary of the poplar MIR171genes. The poplar MIR171gene family expanded mainly through large-scale duplication48-54million years ago and the expression patterns and functions have diversified. MIR171l/m/n have the highest expression activity in many tissues. Poplar MIR171gene family involves in complex regulation of development, light responsiveness and photomorphogenesis by targeting GRAS transcription factors and signal transduction proteins.3. Molecular evolution of the poplar MIR156gene family. The ptc-MIR156gene family expanded mainly via large-scale duplications instead of tandem duplications. The expression patterns and cis-acting elements have diversified among the members in this family and the MIR156g/h/i/j have the highest level of expression. Owing to the variance in the mature sequences, the target genes, including29SBP domain proteins, also differ among these members. These findings suggest that ptc-MIRl56gene family have diversified in functions and formed complex regulatory networks in poplar.4. Evolution of4new miRNA gene families in poplar. The more conserved gene family, MIR1446, expanded mainly through chromosome large-scale duplications, and MIR1444expanded through chromosome large-scale duplications as well as tandem duplications, and the less conserved gene families, MIR6425and MIR6462expanded mainly via tandem duplications; the expression patterns of the4gene families have all diversified, the MIR1444/1446gene families expressed higher in multiple tissues than MIR6425/6462families; the mature sequences of different members are same in MIR1446/6425/6462families, therefore their target genes are same; on the other hand, the mature sequences of different members are different in MIR1444families, so their target genes are different. Comparative analysis of the above-mentioned seven miRNA gene families shows that the older and more conserved miRNA gene families generally contain more members, higher expression levels, higher rates of functional diversification, and their target genes include mainly transcription factors; whereas the younger and less conserved miRNA gene families usually have fewer members, lower expression levels, lower rates of functional diversification, and their target genes are mainly functional protein （e.g., enzymes）.5. Degradome sequencing and gene ontology enrichment analysis of miRNAs target genes in polar. A leaf degradome library of Populus euphratica was constructed and sequenced by Illumina HighSeqTM platform, yielding42,659,177row reads. After removing the adaptors sequences, pollution sequences and low-quality sequences,34,657,804clean reads were obtained. A total of23miRNA targets were identified by analysis of the degradome sequences. All targets identified by degradome analysis were subjected to AgriGO to investigate gene ontology. The miRNA targets were significantly enriched in oxidoreductase activity, antioxidant activity and cell redox homeostasis. These results suggested that miRNAs play important roles in eliminating of ROS and maintaining of redox homeostasis under salt stress. The targets of highly conserved and lowly conserved miRNAs from poplar, Arabidopsis and rice were analyzed by singular enrichment analysis and the realist were cross compared. The results showed that the targets of more conserved miRNAs from above3species were enriched significantly in transcriptional factors, and the targets of more conserved miRNA from poplar were also enriched significantly in oxidation reduction process. In contrast, the enrichment significance of the targets of less conserved miRNAs was lower than more conserved miRNAs。5. Cloning and functional analysis of miRNA target genes in polar. The full length cDNA of one of the MIR171targets, Pescll, was cloned from Populus euphratica. The Pescll belongs to the GRAS transcription factor family and has a miR171a-3p/b target site which was validated by RNA ligase mediated5’RACE. Under salt stress conditions, the expression of Pescll rose in leaves, suggesting that the Pescll participates in response to salt stress in polar. The full length cDNA of Pecngcl, which belongs to the GRAS transcription factor family, was cloned from Populus euphratica. The PeCNGCl located in the cell membrane, which was validated by transient expression in tobacco epidermal cell. The Arabidopsis plants transferred by Pecngcl showed higher salt tolerance than wild type. Under salt stress conditions, the expression of Pecngcl rose in leaves, suggesting that the Pecngcl involved in maintaining of Na+/K+homeostasis in leaves under salt stress in polar.更多还原