本文選題：擬南芥　切入點(diǎn)：花粉管　出處：《中國農(nóng)業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：花粉管的極性生長是保證開花植物雙受精正常完成的必要條件,而這一過程依賴于高度動態(tài)的微絲細(xì)胞骨架。微絲骨架在花粉管中的有序排布和高度動態(tài)受到一系列微絲結(jié)合蛋白的調(diào)控。Profilin是細(xì)胞中含量豐富的微絲結(jié)合蛋白,可以與單體肌動蛋白以1：1的比例結(jié)合,對微絲動態(tài)進(jìn)行雙重調(diào)控。然而profilin在花粉管中是如何對微絲骨架的排布及動態(tài)進(jìn)行調(diào)控的目前尚不清楚,因此本論文以擬南芥花粉中高度特異表達(dá)的PRF4和PRF5作為研究對象,對profilin在花粉管中的功能進(jìn)行深入研究。首先對野生型和profilin功能缺失突變體prf4、prf5及prrf4 prf5花粉管的表型進(jìn)行觀察,結(jié)果發(fā)現(xiàn)profilin突變體花粉的萌發(fā)率與野生型基本相同,而花粉管的生長速率明顯變慢,prf4 prf5雙突變體的花粉管形態(tài)也變得異常。這表明PRF4和PRF5共同參與調(diào)控花粉管的極性生長。為了探究PRF4和PRF5調(diào)控花粉管極性生長的分子機(jī)制,對profilin突變體花粉管中的微絲骨架進(jìn)行固定染色觀察,發(fā)現(xiàn)profilin突變體花粉管頂端和亞頂端的微絲含量明顯減少,微絲排布紊亂。進(jìn)一步在prf4 prf5雙突變體花粉管中引入微絲活體探針Lat52:Lifeact-EGFP,發(fā)現(xiàn)prf4 prf5花粉管頂端質(zhì)膜上雖然有微絲不斷成核,但這些成核的微絲大都不能正常伸長,因此造成亞頂端微絲含量減少,排布紊亂。而亞頂端微絲骨架對于囊泡聚集有重要作用,因此在prf4 prf5雙突變體花粉管中引入囊泡探針Lat52:YFP-RabA4b,發(fā)現(xiàn)prf4 prf5花粉管頂端囊泡的分布范圍變的擴(kuò)散,囊泡的運(yùn)輸速率變慢,花粉管細(xì)胞壁成分發(fā)生改變。這些結(jié)果表明PRF4和PRF5通過促進(jìn)頂端微絲聚合來調(diào)控花粉管的極性生長。本研究選取PRF5作為代表,發(fā)現(xiàn)PRF5與PLP(多聚L-脯氨酸)親和性降低的轉(zhuǎn)基因株系(pgPRF5Y6A;prf4 prf5)花粉管頂端和亞頂端的微絲含量及花粉管的生長速率都沒有恢復(fù)到prf4單突變體水平。此外,體外生化實(shí)驗(yàn)表明富含PLP的formin5能夠利用PRF5-actin復(fù)合體進(jìn)行微絲聚合,但不能有效的利用lPRF5Y6A-actin復(fù)合體。這些結(jié)果表明profilin對微絲聚合的促進(jìn)作用可能是通過formin介導(dǎo)的。借助微絲解聚劑LatB,本研究發(fā)現(xiàn)profilin突變體花粉粒及花粉管對LatB的敏感性明顯低于野生型,這表明profilin能夠促進(jìn)微絲的動態(tài)周轉(zhuǎn)。此外,PRF5與單體肌動蛋白結(jié)合功能缺失的轉(zhuǎn)基因株系(pgPRF5K89E;prf5)花粉管不能互補(bǔ)prf5對LatB不敏感的表型,而PRF5與PLP親和性降低的轉(zhuǎn)基因株系(pgPRF5Y6A;prf5)的花粉管能夠互補(bǔ)。這表明profilin與單體肌動蛋白的結(jié)合對其促進(jìn)微絲動態(tài)周轉(zhuǎn)是重要的,而profilin與PLP的結(jié)合并不影響這一過程。此外,對PRF4和PRF5 N端融合EGFP的轉(zhuǎn)基因植株花粉管進(jìn)行觀察,發(fā)現(xiàn)PRF4和PRF5均勻分布在花粉管細(xì)胞質(zhì)中,這表明profilin-actin復(fù)合體均勻分布在花粉管細(xì)胞質(zhì)中。本論文的研究結(jié)果表明均勻分布在花粉管中的PRF4和PRF5是花粉管極性生長的重要調(diào)控因子。論文在同一個(gè)細(xì)胞中對profilin對微絲動態(tài)的雙重調(diào)控作用進(jìn)行解析,也證明了profilin與單體肌動蛋白的結(jié)合對于其促進(jìn)微絲動態(tài)周轉(zhuǎn)是重要的,而profilin促進(jìn)頂端微絲聚合可能是通過富含PLP的formin介導(dǎo)的,并且這種微絲聚合是花粉管頂端微絲聚合的主要途徑。
[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.

【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q943
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本文編號：1636201