拟南芥短根突变体14-2M-4的表型分析与基因定位分析

发布时间：2018-09-17 15:35

【摘要】：根系是植物重要的地下器官,为植物地上部分提供水分和矿质营养,使其固定在土壤中,并能够根据不断变化的外在环境和内在环境调整其生长,使植物体更好地适应环境。植物根系起源于根尖分生组织中的干细胞,干细胞位于特定的微环境中,通过细胞间的信号交流,维持干细胞活性与特征。根尖干细胞微环境(Root Stem Cell Niche,简称RSCN)由根的静止中心(Quiescent Center,简称QC)和周围的干细胞组成。RSCN结构与功能的稳定对于根系的正常发育至关重要。本研究利用遗传学方法筛选短根突变体,希望找到调控根尖干细胞微环境新的调控元件。本实验室前期工作通过EMS化学诱变的方法建立了突变体库。本研究利用前期获得的短根突变体为材料,将其命名为14-2 M-4,通过对突变体表型的分析和基因的定位分析,获得结果如下:(1)14-2 M-4突变体的根尖分生组织细胞数减少,侧根密度增加,花器官发育异常14-2 M-4突变体的幼苗阶段具有的根系发育缺陷,主要表现为显著的短根,分生组织长度及分生区细胞数显著减少,侧根密度增加。此外,14-2 M-4突变体的花器官发育异常,角果着生位置异常。表明突变的基因影响拟南芥地上部分及地下部分的发育。(2)14-2 M-4突变体中QC表达的报告基因及Cyclin B1;1的表达受抑制通过GUS染色分析,发现在14-2 M-4突变体中RopGEF7等报告基因的表达显著减弱,Cyclin B1;1表达同样减弱,说明突变体中根尖干细胞微环境的维持与分生组织区的细胞分裂活性受到了影响。(3)14-2 M-4突变体中根干细胞决定基因PLT1和PLT2表达减弱在14-2 M-4突变体中SHR与SCR的表达没有受到明显的影响,而PLT1和PLT2的表达则显著减弱,这说明突变的基因影响了PLT的信号通路介导的根尖干细胞微环境的维持。(4)14-2 M-4突变体中PIN1的极性定位改变,改变了生长素分布14-2 M-4突变体中生长素输出载体PIN1在中柱细胞中由底膜定位变为顶膜定位,DR5::GUS在根尖的表达明显减弱,说明突变的基因通过影响PIN1的极性定位,改变了生长素在根尖的分布。(5)基因图位克隆分析确定突变位点在染色体上的区间范围通过遗传学分析表明,突变的基因遗传特性符合孟德尔遗传定律,即分离比为3:1,14-2 M-4突变体属于单基因隐性突变。利用图位克隆技术,把14-2 M-4突变体的突变位点,粗定位于三号染色体长臂上标记NT204附近,通过精细定位,将突变位点定位于MIG5-B和MRC8-B之间,距离约为104kb,区段内共有29个基因。综上所述,14-2 M-4突变体中基因突变导致了根系的发育缺陷,影响生长素介导的器官形成。因此,该基因可能在生长素调控根系发育的分子网络中起重要作用。
[Abstract]:Root system is an important underground organ of plants, which provides water and mineral nutrients for the aboveground parts of plants, makes them fixed in soil, and adjusts their growth according to the changing external and internal environment, so that the plants can adapt to the environment better. Plant roots originated from stem cells in root tip meristem cells. Stem cells are located in specific microenvironments and maintain stem cell activity and characteristics through the communication of signals between cells. Root tip stem cell microenvironment (RSCN) is composed of the rest center of root (Quiescent Center, (QC) and the surrounding stem cells. The stability of structure and function of RSCN is very important for the normal development of root system. In this study, genetic methods were used to screen short root mutants in order to find new regulatory elements to regulate the microenvironment of root tip stem cells. The mutant library was established by EMS chemical mutagenesis. In this study, the short root mutants were used as materials and named 14-2 M-4. The results were as follows: (1) the number of root meristem cells in 14-2 M-4 mutants decreased, and the density of lateral roots increased. In the seedling stage of the mutant 14-2 M-4, the defects of root development were mainly short roots, the length of meristem and the number of meristem cells decreased significantly, and the density of lateral roots increased. In addition, the floral organs of the mutants 14-2-M-4 were abnormal, and the position of pods was abnormal. The results showed that the mutant gene affected the development of the aboveground and underground parts of Arabidopsis thaliana. (2) the reporter gene of QC expression and the expression of Cyclin B1O1 in 14-2 M-4 mutant were inhibited by GUS staining. It was found that the expression of RopGEF7 and other reporter genes was significantly decreased in 14-2 M-4 mutant. These results indicate that the maintenance of microenvironment of root tip stem cells and the mitotic activity of meristem cells in mutants are affected. (3) the expression of PLT1 and PLT2 of root stem cell determinants in 14-2 M-4 mutants is weakened in 14-2 M-4 mutants, and the expression of SHR and SCR in 14-2 M-4 mutants is not found. Has been significantly affected, However, the expression of PLT1 and PLT2 decreased significantly, which suggested that the mutant gene affected the maintenance of the microenvironment of root stem cells mediated by the signal pathway of PLT. (4) the polar localization of PIN1 in the 14-2 M-4 mutant was changed. The expression of auxin output vector PIN1 in the mutant 14-2 M-4 was significantly decreased in the medium column cells from the bottom membrane localization to the apical membrane localization, indicating that the mutant gene affected the polarity of PIN1 by affecting the expression of DR5: Gus in the root tip. The distribution of auxin in the root tip was changed. (5) genetic mapping and cloning analysis confirmed the range of the mutation loci on the chromosome. Genetic analysis showed that the genetic characteristics of the mutation were in accordance with Mendelian's law of heredity. The isolation ratio of the mutant was 3: 1, 14-2, M-4, which belonged to the single gene recessive mutation. The mutational sites of 14-2 M-4 mutants were mapped coarsely near the NT204 labeled on the long arm of chromosome 3 by map cloning technique. By fine mapping, the mutation sites were located between MIG5-B and MRC8-B, the distance was about 104 kb, and there were 29 genes in the region. In conclusion, the gene mutation in the 14-2 M-4 mutant resulted in defects in root development and affected auxin mediated organ formation. Therefore, this gene may play an important role in the molecular network of auxin regulating root development.
【学位授予单位】：华南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2

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