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Functional Characterization Of The Arabidopsis AtHMGB15Gene In Pollen Tube Germination And Growth

Posted on:2015-06-21Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y J WangFull Text:PDF
GTID:1220330467950305Subject:Cell biology
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In flowering plants, Arabidopsis male gametes (sperm cells) develop wthin male gametophytes (pollen grains). A mature pollen grain is a three-cell life unit that consists of one vegetative cell in which two sperm cells are enclosed. During anthesis, the pollen grains are pollinated to the stigma of the pistil. After recognification with the stigmatic cells, they hydrates and then generates a pollen tube. The pollen tube growth is a polarized elongation envent in the transmitting tract towards a female gametophyte (embryo sac) and delivers the sperm cells into the embryo sac for double fertilization. Therefore, pollen germination and pollen tube growth are crucial for plant sexual reproduction. However, the mechanisms of the gene transcription regulation during pollen germination and pollen tube growth remain poorly understood.Previously, our research group identified the transcription factor gene AtHMGB15that played an important role in the pollen tube germination and growth using Ac/Ds gene grap and enhancer grap method. The preliminary results showed that AtHMGB15gene was specifically expressed in pollen and pollen tube and the AtHMGB15protein was localized in the vegetative nucleus of pollen. The athmgb15-1mutant exhibited significantly reduced germination rate and genetic transmission efficiency. In this study, we further studied the function and regulatory mechanism of AtHMGB15gene by intensively characterizing the phenotype and genetic characteristic of the athmgbl5-1mutant, the expression pattern of AtHMGB15gene, and the function of AtHMGB15protein. The results showed that the self-pollinated athmgb15-1plants had much lower seed set and short siliques, compared to those in the wild type. The manual pollination showed that most of the athmgb15-1pollen grains could germinate. However, the resulting pollen tubes were short and unable to reach the embryo sac, disrupting the fertilization. AtHMGB15encodes a protein that belongs to the plant-specific ARID-HMG family, whose amino acid sequence shares a high similarility with ARID-HMG1, ARID-HMG2and AtHMGB11. Genetic analysis showed that mutations in ARID-HMG1, ARID-HMG2or AtHMGB11did not affect the function of male gametes, respectively. A double mutant in AtHMGB11and AtHMGB15had an enhanced phenotype compared to the athmgb15-1mutant, suggesting that AtHMGBll and AtHMGB15may be functionally redundant.AtHMGB15protein has the ARID and HMG-box domains like the other ARID-HMG proteins. It is able to specifically bind to the special DNA structure in vitro and form homomultimers with itself or heteromultimers with other ARID-HMG proteins. Transcription microarray analysis using mature pollen grains showed that there were about1686genes whose expression was altered by more than2-folds in the athmgb15-1mutant compared to wild type, among which585genes were downregulated and1101genes were upregulated, including those involved in cell wall synthesis and modification, signaling, vesicle transport, transcription and translation, etc. Many of the altered genes have been identified as being important for pollen development and pollen tube germination growth. Bimolecular fluorescence complementation and luciferase complementation assays showed that AtHMGB15could interact with the pollen transcription factors AGL66and AGL104. A comparison of the transcription profile of the athmgb15-1and ag166agl104-2mutant showed that AtHMGB15, AGL66and AGL104may co-regulate the transcription of many genes in pollen. Genetic analysis showed that the athmgbl5-1ag166agl104-2triple mutant had an enhanced phenotype compared to the athmgbl5-1single mutant and ag166ag1104-2double mutant. These results suggest that AtHMGB15, AGL66and AGL104are likely to function in the same pathway. Furthermore, AtHMGB15could interact with the general transcription factor AtTFIIB1and the MADS transcription factor AGL18.In summary, AtHMGB15may function as a homomultimer and control pollen germination and pollen tube growth through regulating the expression of the down stream genes by interaction with other transcription factors AGL66, AGL104, AGL18and AtTFIIB1.
Keywords/Search Tags:Arabidopsis thaliana, transcription factor, ARID-HMG, pollen tube germination andgrowth
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