【作者】 唐芳；

【导师】 卢孟柱；

【作者基本信息】 中国林业科学研究院 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 木材形成是一个复杂的生物学过程,是维管形成层生长和分化出次生维管组织的结果。植物的次生生长是一个高度有序的细胞分化、发育过程,由调控基因参与调控的许多功能基因协同表达保证这一过程的有序进行。microRNA(miRNA)是一类内源性、由非编码基因转录的单链小RNA,是真核生物基因表达中的一类负调控因子,主要在转录后水平上通过介导靶基因mRNA的切割或抑制翻译来调节植物基因的表达。为研究miRNAs在次生维管系统形成过程中的调控作用,本论文以毛白杨为研究对象,利用前期建立的剥皮维管再生实验系统为基础,通过μParaflo? microRNA微阵列芯片分析了毛白杨维管再生过程中miRNAs的差异表达谱。对毛白杨维管再生过程中第0、6、8、10、12、14、16、18、20和22天前后两次连续样品间miRNAs表达的差异分析,得到了差异表达的99个miRNA,其中大部分来自于miRNA159、miRNA164、miRNA166、miRNA319、miRNA396、miRNA168和miRNA399七个家族。聚类分析表明,在维管再生过程中表达模式发生明显变化的72个miRNA,大部分都是以上七个miRNA家族的成员,说明这七个miRNA家族在维管再生过程中起到重要的调控功能。它们所调控的靶基因多为转录因子家族的成员,可能参与调控毛白杨形成层发生、发育和分化以及木质部的发育。杨树剥皮后再生过程的第12、14、16、18、22天miRNA的表达变化较大,因此,这几个时期可能是次生维管形成的关键发育转换时期。定量PCR法验证了部分miRNA的表达。根据miRNA的靶基因家族与其在ATH1芯片中的表达情况,选择在次生维管再生后期特异表达且在木质部表达量高的MYB转录因子家族为研究对象。选取该家族中6个基因的7个拟南芥T-DNA插入突变系,在8小时短日照的条件下次生诱导培养,成功筛选到与野生型茎基部次生木质部产生很大变化的突变体,命名为CWT。CWT纯合突变植株的茎基部维管束间部位次生木质部的细胞数量减少、细胞壁明显增厚,该部位各层细胞的次生壁都有程度均一的增厚,周皮和髓位置的细胞壁没有加厚,不同于野生型植株在该位置向心方向的细胞壁增厚程度逐渐减小。将得到的纯合突变T2代种子继续种植,T3代具有稳定的遗传突变性状,因没有得到杂合体,突变产生的性状也没有发生分离。本研究通过miRNA芯片分析得到了可能参与调控次生维管形成过程的7个miRNA家族成员,初步确定了它们参与次生维管形成的关键发育时期。通过对miRNA159靶基因MYB转录因子家族中部分基因的拟南芥突变体的表型研究,证实MYB基因对维管次生生长有相关作用。为进一步分析miRNA和其靶基因的表达和功能,揭示两者的相互调控关系奠定基础,并为材性改良的基因工程育种提供理论依据。更多还原

【Abstract】 Wood formation is a complex progress composing many biological events,occurs as a result of growth and differentiation of the vascular cambium. This secondary growth is a highly ordered developmental progress,which coordinates functional genes and different regulators. MicroRNAs (miRNAs), a group of small non-coding RNAs, are a class of post-transcriptional negative regulators playing vital roles in plant development and growth. To investigate miRNAs involved in secondary vascular system (SVS) development, aμParaflo? MicroRNA microarray was used to analyze miRNAs expression profiles during regeneration of SVS in Chinese white poplar (Populus tomentosa).By comparing the expressional profiles of adjacent regeneration stages, 99 differentially expressed miRNAs were identified, most of them belong to the superfamilies of miRNA159, miRNA164, miRNA166, miRNA319, miRNA396, miRNA168 and miRNA399. Cluster analysis showed that the expression pattern of 76 miRNAs changed dramatically during SVS formation. Those miRNAs also belong to the seven miRNAs superfamilies mentioned above. Previous data proved that the targets of these miRNAs are transcriptional factors play important roles in wood formation. Thus, we can suggest that some members of these miRNA families might be the negative regulators of the SVS in poplar. And those expressed differentially on 12th, 14th, 16th and 22nd day of the regeneration might involve in the stage switches during SVS regeneration. The expression pattern of several miRNAs was confirmed by real time quantitive PCR.To characterize the function of miRNA target genes, 7 Arabidopsis T-DNA mutant lines of MYB were chosen to further analysis according to the previous data on their differentially expression during SVS in poplar. The mutants and wild type(WT) Arabidopsis were cultured under secondary growth inducing condition. An Arabidopsis mutant named CWT had obviously changes in interfascicular regions, where the secondary walls of homozygous cwt mutant were clearly thickened, the cell cavity and quantity of interfascicular fiber reduced relatively. Secondary walls of xylem thickened equally but gradually in WT lines.miRNAs profiling during SVS regeneration were performed by liquid miRNAs chip in this study. The results showed that several miRNAs might play important roles in cambium development and xylem differentiation. And mutant analysis of MYB, the target gene of miRNA159, showed the irregular cell wall thickness in certain region, which suggested this miRNA might regulate the formation of secondary wall. Our data might provide new insight into the transcriptional regulatory networks of wood formation, and select candidate genes may be responsible for the traits of wood property. To isolate further and characterize these key genes is needed to understand their biological function in wood formation in a molecular breeding point of view.更多还原