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標題: 百合花粉聚半乳醣醛酸酵素基因和蛋白質之特性分析
Characterization of pollen-specific polygalacturonase genes and their proteins in Lilium longiflorum
作者: 江晉毅
Chiang, JinYi
關鍵字: lily;百合;pollen;polygalacturonase;花粉;聚半乳醣醛酸酵素
出版社: 生物科技學研究所
聚半乳醣醛酸酵素(polygalacturonase; PG)屬於醣基水解酶(glycosyl hydrolase family 28)的成員,其參與果實成熟、組織離層、創傷與防禦反應、花粉的成熟和花粉管生長等過程。本實驗主要研究鐵砲百合(Lilium longiflorum)花粉聚半乳醣醛酸酵素基因和蛋白質的特性。利用5’-和3’-RACE(rapid amplification of cDNA ends)方法獲得一個421 bp的5’-RACE片段及兩個分別為443 bp和505 bp(不含poly(A))的3’-RACE片段。序列分析得知,百合花粉聚半乳醣醛酸酵素基因的轉譯區包含1242 bp可轉譯出413個氨基酸。百合花粉聚半乳醣醛酸酵素基因具有兩個插入子(intron),其大小分別是113 bp和105 bp。其預測氨基酸序列的N端有一段為24個氨基酸的疏水性訊息胜肽(signal peptide)。以氨基酸序列比對後,百合與其他植物花粉聚半乳醣醛酸酵素有40∼50 %的相同度(identity),並且具有四個高度保留的區域,此區域與酵素活性有關。為了取得抗百合花粉聚半乳醣醛酸酵素抗體,利用大腸桿菌BL21大量表現百合花粉聚半乳醣醛酸酵素融合蛋白,以Ni-NTA瓊膠純化,再施打於大鼠體內而得到抗百合花粉聚半乳醣醛酸酵素抗血清。以轉漬親和(blot-affinity)方式純化抗百合花粉聚半乳醣醛酸酵素抗體,進行2D-Western blotting分析顯示百合花粉聚半乳醣醛酸酵素蛋白為多型性(polymorphism)的醣蛋白,具有分子量和pI值的差異性。百合花粉聚半乳醣醛酸酵素蛋白具有花粉專一性,於花粉成熟晚期開始合成。由未成熟的先行乾燥實驗可知百合花粉聚半乳醣醛酸酵素蛋白並不會受到乾燥所誘導而產生。花粉萌發一段時間之後,仍然可偵測到百合花粉聚半乳醣醛酸酵素蛋白,推測百合花粉聚半乳醣醛酸酵素蛋白可能與花粉萌發及花粉管生長有關。百合花粉聚半乳醣醛酸酵素蛋白可被貓尾草Phl p 13過敏原的抗體認識,但有過敏反應的病人血清並不認識百合花粉蛋白質。

Polygalacturonase (PG) belongs to a family 28 of glycosyl hydrolases and is involved in fruit ripening, tissure abscission, wound and defense response, pollen maturation and pollen tube growth. In this study, we have characterized PG genes and their proteins in Lilium longiflorum pollen. Using 5'-and 3'-RACE (rapid amplification of cDNA ends) analysis, a 5'-RACE fragment (421bp) and two 3'-RACE fragments (465 and 521 bp, excluding the 3' poly(A) tail) were obtained. Sequence analysis revealed that lily PG gene contains a coding region of 1242 bp encoding 413 amino acids. The coding region of the gene is interrupted by two introns of 113 bp and 105 bp, respectively. The predicted amino acid sequence of lily PG sequence has an N-terminal hydrophobic signal peptide of 24 amino acids. Comparison of lily pollen PG with others shows 40~50 % sequence identity and exhibits four conserved domains involved in the catalytic reaction. To obtain anti-lily PG antiserum, the coding region of lily PG cDNA was introduced into pET32a and expressed in Escherichia coli BL21 from which the fusion protein was isolated and purified by Ni-NTA agarose. Using affinity-purified lily PG antibody, 2D-Western blot analysis indicated that lily PG protein has a polymorphic form, displaying differences in molecular weight and pI. Immunoblots indicated that lily PG protein is probably a high mannose glycoprotein. The protein is pollen-specific and accumulates only at the later stage of pollen maturation. Premature drying analysis suggested that the lily PG protein was not induced by desiccation. When germinated, the lily PG protein remained its level for a long while before it was degraded during pollen tube growth. It suggests that the protein may be involved in pollen germination and pollen tube growth. While anti-Phl p 13 Ab recognized lily PG protein, the patient serum allergic to Phl p 13 did not recognize lily PG protein.
Appears in Collections:生物科技學研究所

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