拟南芥ROP6信号通路调控细胞极性发育的突变体筛选和突变基因克隆

发布时间：2018-05-18 14:02

本文选题：细胞极性 + AN　；参考：《福建农林大学》2017年硕士论文

【摘要】：细胞极性,广义上是细胞内的不对称分布,是细胞实现发育和环境调节功能的基本特征之一。拟南芥作为模式植物,其单细胞的花粉管和多细胞表皮系统已经成为研究植物细胞极性的重要模式系统。早先有研究表明在拟南芥的子叶表皮细胞中,生长素可以激活植物细胞极性通路下游特有的ROP6(Rho-related GTPase from plants 6)-RIC1(ROP-interactive CRIB motif-containing protein 1)信号转导途径,进而影响细胞骨架的排列来调控细胞极性生长和形态建成。ROP6-RIC1信号途径可以促进细胞凹陷区(Neck Region)周质微管的有序排列,进而维持细胞的生长极性。本论文通过正向遗传学方法筛选ROP6-RIC1途径中的新成员。在rop6-2缺失背景下,本实验通过EMS(ethylmethane-sulfonate,甲基磺酸乙酯)诱变筛选拟南芥表皮铺板细胞中凸起数量(Lobe Number)明显降低的突变体—507-2。经遗传学方法验证该突变是一个隐性纯和的突变体。通过BSA-重测序(Bulked Sergeant Analysis)方法找到了可能导致该突变的四个候选基因。在对四个候选基因进行鉴定分析后,确认突变基因为Angustifolia(AN)。突变发生于AN基因上的第877脱氧核糖核酸上,其中的G突变成了 A并导致其编码的第293个氨基酸谷氨酸(E)突变为赖氨酸(K)。根据突变基因的特点,将去掉rop6-2背景的单基因突变体507-2-SM命名为ANE293K。之前的研究证实AN在细胞质,细胞核以及高尔基体等上表达。本实验用烟草做AN和ANE293K的瞬时表达,发现其在细胞质以及类似片层结构的高尔基体上皆有表达。AN和AN E293K蛋白在细胞内都能运动,但是AN的运动速率明显高于ANE293K。观察ANE293K的微管排列分布,发现较野生型相比,AN E293K的微管束较粗且其分布密度较大,说明突变的AN影响到微管的组织动态。而且在ANE293K里也没有观察到在野生型里面垂直于细胞生长方向抑制细胞生长的微管束。507-2的细胞表型与ANE293K更为相似,因此AN是位于ROP6信号通路途径的下游来调控细胞极性生长,但具体调控机制尚需进一步实验进行解析。由于AN和ANE293K在野生型和突变体里面的转录水平没有发生变化,本论文推测是ANE293K蛋白生化功能上的改变,导致其影响了细胞微管的组织动态并最终影响其细胞的极性建成。
[Abstract]:Cell polarity, in a broad sense, is the asymmetric distribution of cells, which is one of the basic characteristics of cell development and environmental regulation. Arabidopsis thaliana (Arabidopsis thaliana) as a model plant, its single cell pollen tube and multicellular epidermal system have become an important model system for studying the polarity of plant cells. Previous studies have shown that in Arabidopsis thaliana cotyledon epidermis cells, auxin activates the ROP6(Rho-related GTPase from plants 6)-RIC1(ROP-interactive CRIB motif-containing protein 1) signal transduction pathway that is unique downstream of the plant cell polarity pathway. Furthermore, the regulation of cell polarity growth and morphogenesis by affecting the arrangement of cytoskeleton. ROP6-RIC1 signaling pathway can promote the ordered arrangement of periplasmic microtubules in the cell depression area check region, and then maintain the growth polarity of the cells. In this paper, the new members of ROP6-RIC1 pathway were screened by forward genetics. Under the background of rop6-2 deletion, the mutant -507-2was screened by mutagenesis of ethylmethane-sulfonate (ethyl methane-sulfonate) in Arabidopsis thaliana epidermal paving cells. The mutation was proved to be a recessive pure sum mutant by genetic methods. Four candidate genes for the mutation were identified by BSA-re sequencing and barked Sergeant analysis. After identification and analysis of four candidate genes, it was confirmed that the mutant gene was Angustifolia angustifolia. The mutation occurred in the 877 deoxyribonucleic acid (DNA) of an gene, in which the G process became A and led to the mutation of the 293rd amino acid glutamate E) to Lysine Ko. According to the characteristics of the mutant gene, the single gene mutant 507-2-SM, which removed the background of rop6-2, was named ANE293K. Previous studies have confirmed an expression in cytoplasm, nucleus and Golgi apparatus. In this study, tobacco was used for transient expression of an and ANE293K. It was found that both an and an E293K proteins could be expressed in the cytoplasm and Golgi bodies similar to lamellar structure, but the movement rate of an was significantly higher than that of ANE293K. The distribution of microtubules in ANE293K was observed. The results showed that the microtubule bundles of ANE293K were thicker and denser than those of wild type, indicating that the mutant an affected the tissue dynamics of microtubules. Moreover, the phenotypes of microtubules .507-2, which are perpendicular to the growth direction of cells, were not observed in ANE293K, and the phenotype of microtubules .507-2 was more similar to that of ANE293K, so an is located downstream of the ROP6 signaling pathway to regulate cell polar growth. However, the specific regulatory mechanisms need further experimental analysis. Since the transcription levels of an and ANE293K in wild type and mutant have not changed, this paper speculated that the changes of biochemical function of ANE293K protein resulted in its influence on the cellular microtubule tissue dynamics and ultimately on the cell polarity formation.
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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