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標題: 百合花粉中與乾燥相關蛋白質LLA-32之免疫金粒標定及萌發相關基因之研究篩選
mmunolocalization of Desiccation Associated LLA-32 Protein and Characterization of Pollen Germination Associated Genes in Lily Pollen
作者: 蘇庭禾
Su, Ting-Ho
關鍵字: Lily
Pollen Germination
monosaccharide transporter
出版社: 農業生物科技學研究所
摘要: LLA-32是一個與百合花粉乾燥有關的蛋白質,表現於花粉發育的最後一個階段。在之前的研究中已知花粉管內LLA-32的含量會隨萌發時間的增加而逐漸減少。為了要進一步研究LLA-32在花粉發育後期及花粉管生長時表現的位置及可能扮演之角色,我們利用親和性純化之LLA-32抗體進行免疫金粒標定。實驗結果顯示,LLA-32主要表現於花粉外壁(exine),可能來自花藥絨氈層。另外也存於營養細胞核、生殖細胞核、生殖細胞質中,但在營養細胞質及花粉管其含量較少,於花粉粒內壁(intine)則幾乎無標記。目前推測其於花粉內的功用可能為保護花粉粒,使花粉粒在萌發前不受乾燥的侵害。 低溫儲存過的百合花粉萌發及生長速率會比新鮮花粉低,為了探討這個現象,在之前的研究中分別抽出儲存及新鮮花粉的全RNA,利用supression subtractive hybridization的方式,找出於低溫儲存過程中一些mRNA被降解的的基因,本論文中發現了45個基因片段;此外本實驗室也以上述方式發現63個基因片段,其中一個為單醣運輸蛋白質(LLPD22)。本論文以5'及3'-RACE的方式得到其cDNA全長共計1851 nt,並可轉譯出含505個氨基酸之蛋白質,分子量55.0 kDa,pI值為6.4。單醣運輸蛋白質在醣類運輸、轉換和能量產生的機制中有著重要地位。目前推測花粉管壁膜上之轉化酵素(invertase)可將蔗糖分解成果糖和葡萄糖,再經由單醣運輸蛋白質把果糖和葡萄糖由花粉管外送入花粉管內,以提供花粉管生長所需之能量及構築細胞結構之來源。
LLA-32 is a stress-induced protein and is in association with desiccation in lily (Lilium longiflorum) pollen during the later stage of maturation. Previous study indicated that the accumulation of LLA-32 protein remained steady in the mature pollen, but it was gradually disappeared during pollen tube germination. To further investigate the possible function of LLA-32 during pollen maturation and pollen tube germination, immunolocalization with affinity-purified LLA-32 antisera was performed. Experimental results demonstrated that LLA-32 mainly localized in the pollen outer wall(exine), cytoplasm and nucleus of generative cell, and vegetative nucleus. Less labeling was found in vegetative cytoplasm and germination pollen tube, and was rare in the pollen inner wall(intine). The possible function of LLA-32 in mature lily pollen is protection of the pollen grain before pollen tube germination during desiccation. The germination and growth rates of lily pollen grain are dramatically reduced after low temperature storage. By using supression subtractive hybridizatio, the mRNAs degraded during -20℃ storage were isolated. In this studying, 45 cDNA clones were obtained. In the parallel experiment, one cDNA encoded for the monosaccharide transporter(LLPD22)obtained and chosen for further characterization. To obtain its full length, 5'- and 3'-RACE were perform. One full length of 1851 nucleotides was obtained and contained one opening reading with 505 a.a. Its estimated molecular weight and pI are 55 kDa and 6.4, respectively. It has been suggested that monosaccharide transporters play critical rules in sugars transport across plasma membrane. The invertase of pollen tube membranes may convert the sucrose into fructose and glucose, which are transported by monosaccharide transporters into pollen tubes to provide the energy and cell skeleton source during pollen tube growth.
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