拟南芥profilin在花粉管生长过程中的功能和作用机制的研究

发布时间：2018-03-19 22:15

  本文选题：拟南芥　切入点：花粉管　出处：《中国农业大学》2016年博士论文　论文类型：学位论文

【摘要】：花粉管的极性生长是保证开花植物双受精正常完成的必要条件,而这一过程依赖于高度动态的微丝细胞骨架。微丝骨架在花粉管中的有序排布和高度动态受到一系列微丝结合蛋白的调控。Profilin是细胞中含量丰富的微丝结合蛋白,可以与单体肌动蛋白以1：1的比例结合,对微丝动态进行双重调控。然而profilin在花粉管中是如何对微丝骨架的排布及动态进行调控的目前尚不清楚,因此本论文以拟南芥花粉中高度特异表达的PRF4和PRF5作为研究对象,对profilin在花粉管中的功能进行深入研究。首先对野生型和profilin功能缺失突变体prf4、prf5及prrf4 prf5花粉管的表型进行观察,结果发现profilin突变体花粉的萌发率与野生型基本相同,而花粉管的生长速率明显变慢,prf4 prf5双突变体的花粉管形态也变得异常。这表明PRF4和PRF5共同参与调控花粉管的极性生长。为了探究PRF4和PRF5调控花粉管极性生长的分子机制,对profilin突变体花粉管中的微丝骨架进行固定染色观察,发现profilin突变体花粉管顶端和亚顶端的微丝含量明显减少,微丝排布紊乱。进一步在prf4 prf5双突变体花粉管中引入微丝活体探针Lat52:Lifeact-EGFP,发现prf4 prf5花粉管顶端质膜上虽然有微丝不断成核,但这些成核的微丝大都不能正常伸长,因此造成亚顶端微丝含量减少,排布紊乱。而亚顶端微丝骨架对于囊泡聚集有重要作用,因此在prf4 prf5双突变体花粉管中引入囊泡探针Lat52:YFP-RabA4b,发现prf4 prf5花粉管顶端囊泡的分布范围变的扩散,囊泡的运输速率变慢,花粉管细胞壁成分发生改变。这些结果表明PRF4和PRF5通过促进顶端微丝聚合来调控花粉管的极性生长。本研究选取PRF5作为代表,发现PRF5与PLP(多聚L-脯氨酸)亲和性降低的转基因株系(pgPRF5Y6A;prf4 prf5)花粉管顶端和亚顶端的微丝含量及花粉管的生长速率都没有恢复到prf4单突变体水平。此外,体外生化实验表明富含PLP的formin5能够利用PRF5-actin复合体进行微丝聚合,但不能有效的利用lPRF5Y6A-actin复合体。这些结果表明profilin对微丝聚合的促进作用可能是通过formin介导的。借助微丝解聚剂LatB,本研究发现profilin突变体花粉粒及花粉管对LatB的敏感性明显低于野生型,这表明profilin能够促进微丝的动态周转。此外,PRF5与单体肌动蛋白结合功能缺失的转基因株系(pgPRF5K89E;prf5)花粉管不能互补prf5对LatB不敏感的表型,而PRF5与PLP亲和性降低的转基因株系(pgPRF5Y6A;prf5)的花粉管能够互补。这表明profilin与单体肌动蛋白的结合对其促进微丝动态周转是重要的,而profilin与PLP的结合并不影响这一过程。此外,对PRF4和PRF5 N端融合EGFP的转基因植株花粉管进行观察,发现PRF4和PRF5均匀分布在花粉管细胞质中,这表明profilin-actin复合体均匀分布在花粉管细胞质中。本论文的研究结果表明均匀分布在花粉管中的PRF4和PRF5是花粉管极性生长的重要调控因子。论文在同一个细胞中对profilin对微丝动态的双重调控作用进行解析,也证明了profilin与单体肌动蛋白的结合对于其促进微丝动态周转是重要的,而profilin促进顶端微丝聚合可能是通过富含PLP的formin介导的,并且这种微丝聚合是花粉管顶端微丝聚合的主要途径。
[Abstract]:The polar growth of pollen tube is the necessary condition to ensure the normal completion of double fertilization in flowering plants, the microfilament cytoskeleton and this process depends on the highly dynamic actin cytoskeleton. Orderly arrangement in the tubes and by a series of highly dynamic actin binding regulation of.Profilin protein is rich in actin binding protein in the cells, and monomer the ratio of 1:1 to actin binding, dual regulation of actin dynamics. However, profilin in the pollen tube is how the actin cytoskeleton arrangement and dynamic regulation is not clear, so the expression of highly specific Arabidopsis pollen in the PRF4 and PRF5 as the research object, in-depth study of the function of profilin in the pollen tube first of wild type and profilin mutant of prf4, PRF5 and prrf4 PRF5 phenotype of pollen tube were observed. The results showed that profilin The germination rate of wild type and mutant pollen is basically the same, but the growth rate of pollen tubes was slow, prf4 PRF5 double mutant pollen tube morphology becomes abnormal. This indicates that the polar growth of PRF4 and PRF5 participate in the regulation of pollen. In order to explore the PRF4 and PRF5 regulation of polar growth of pollen tubes molecular mechanism, fixed staining of microfilaments in pollen tubes of profilin mutant, profilin mutant actin content found apical and sub apical microfilament arrangement disorder significantly reduced. Further introduction of micro wire probe in prf4 Lat52:Lifeact-EGFP in PRF5 double mutant pollen tube, prf4 PRF5 found that although the apical membrane microfilament continuous nucleation, but these are not normal actin nucleation it resulted in the decrease of elongation, sub apical microfilament content arrangement disorder. And the apex of actin cytoskeleton for vesicle aggregation An important role, so the introduction of vesicles in prf4 probe Lat52:YFP-RabA4b PRF5 double mutant pollen tubes, identified the distribution range of prf4 PRF5 apical vesicles change, vesicle transport rate is slow, the pollen tube cell wall components changed. These results show that PRF4 and PRF5 can promote the apical microfilament polymerization to regulate the polar growth of pollen tube the PRF5 is chosen as the representative. In this study, found that PRF5 and PLP (poly L- proline) reduced the affinity of transgenic lines (pgPRF5Y6A; prf4 PRF5) growth rate of actin content and apical and sub apical pollen tubes are not restored to the prf4 single mutant. In addition, the in vitro biochemical experiments show that PLP rich formin5 for microfilament polymerization using PRF5-actin complex, but can not effectively use lPRF5Y6A-actin complex. The results indicated that profilin of microfilament polymerization promoting effect May be mediated by formin. With the help of latrunculin-A LatB, this study found that the sensitivity of profilin mutant pollen and pollen tube of LatB were significantly lower than that of the wild type, suggesting that dynamic profilin can promote actin turnover. In addition, PRF5 and monomeric actin binding function deficient transgenic lines (pgPRF5K89E; PRF5) tube can not complementary PRF5 is not sensitive to the phenotype of LatB, PRF5 and PLP reduced the affinity of transgenic lines (pgPRF5Y6A; PRF5) the pollen tube can be complementary. This indicates that the combination of profilin and actin monomers are important to promote the dynamic microfilament turnover, and the combination of profilin and PLP did not affect this process. In addition, GM the plants of PRF4 and PRF5 N tube end fusion EGFP were observed, PRF4 and PRF5 are uniformly distributed in the pollen tube cytoplasm, suggesting that the profilin-actin complex is evenly distributed in the pollen Tube in the cytoplasm. The results of this study show that the uniform distribution in the tubes of PRF4 and PRF5 is an important regulatory factor in polar growth of pollen tubes. This paper analyzes the dual role of profilin in regulation of actin dynamics in the same cell, it is proved that the profilin with a single muscle combined with dynamic protein is important for the promotion of profilin and dynamic microfilament turnover, promoting apical microfilament polymerization may be mediated by formin with PLP guide, and the microfilament polymerization is the main way to apical microfilament polymerization.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q943
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本文编号：1636201