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標題: 兩個百合花粉專一cDNAs之特性分析
Characterization of two pollen-specific cDNAs
作者: 黃麗曰
Huang, Li-Yueh
關鍵字: Lilium longiflorum;鐵砲百合;pollen-specific;pollen gene;late gene;花粉專一性;花粉基因;後期基因
出版社: 農業生物科技學研究所
我們由百合成熟花粉的cDNA集合庫得到兩個具花粉專一的cDNA clones。北方墨點分析顯示LLP12-2及LLP12-3 transcripts只在花粉中偵測到,於花絲、雌蕊、花被、根、莖及葉皆無偵測到。而且,LLP12-2和LLP12-3 mRNA的表現,只出現在花粉成熟期,隨著花粉/花藥的成長而有逐漸增加的趨勢。核酸序列分析得知LLP12-2 cDNA的長度為975 bp,LLP12-3為568 bp。北方墨點法分析得知LLP12-2及LLP12-3 mRNA的長度均1,400 核甘酸左右,顯然所得到兩者的cDNA並非全長。利用5’-RACE-PCR得到LLP12-2 1191 bp全長的cDNA。此LLP12-2 cDNA包含319 bp的5’端非轉譯區、可轉譯222個氨基酸669 bp的轉譯區及 203 bp的3’端非轉譯區。LLP12-2所轉譯的氨基酸序列比對後,發現N端有一段序列與數個擬南芥蛋白有47∼59 % 的相同度。LLP12-2蛋白之氨基酸的組成以serine、glycine及proline含量較多,估計的分子量為24 kDa,等電點為9.1,為一親水性蛋白質。可能具有一個α-helix及兩個β-sheets。利用E. coli 大量表達LLP12-2重組蛋白,其分子量約52 kDa。利用5’-RACE-PCR,我們亦得到907 bp的LLP12-3 cDNA。此LLP12-3 cDNA仍然不是全長,其包含一個不完整的open reading frame可譯出199個氨基酸及305 bp的3’端非轉譯區。將LLP12-3轉譯蛋白比對後,發現與擬南芥一蛋白有41 % 的相同度。LLP12-3現有的蛋白部份為親疏水性所交錯,可能具有六個α-helix及三個β-sheets。

Two lily pollen-specific clones identified from a cDNA library made with poly(A) RNA were characterized. The organ-specificity and temporal expression during anther development for LLP12-2 and -3 were analyzed by Northern blot hybridization. It indicated that no mRNA was detected in filaments, pistils, tepals, roots, stems and leaves. In addition, the mRNA levels of LLP12-2 and -3 were gradually accumulated to higher levels at the stage of pollen maturation. After sequencing, the length of LLP12-2 cDNA is 975 bp and that of LLP12-3 cDNA is 568 bp. Northern hybridization revealed that their corresponding mRNAs are 1400 bases, respectively so that apparently the two cDNA are not full length. By the use of 5'-RACE-PCR, a full length of LLP12-2 cDNA was obtained. The LLP12-2 cDNA has 1191 bp, consisting of a 319 bp 5'-untranslated region, an open reading frame of 669 bp encoding 222 amino acids, and a 218 bp 3'-untranslated region. Sequence analysis of the N-terminal region of deduced LLP12-2 protein exhibits significant similarity of the protein to a number of proteins in Arabidopsis, with identities ranging from 47~59 %. The predicted LLP12-2 protein has a calculated molecular mass of 24 kDa and an isoelectric point of 9.1. The secondary structure of LLP12-2 may contain an α-helix, and twoβ-sheets. The LLP12-2 cDNA clone was expressed in E. coli, producing a fused product of 52 kDa protein. Coversely, a 907 bp of LLP12-3 was obtained using 5'-RACE-PCR. The LLP12-3 cDNA contains an incomplete open reading frame of 600 bp encoding 199 amino acids, and a 305 bp 3'-untranslated region. Sequence analysis of partial LLP12-3 protein exhibits significant similarity of the protein to an Arabidopsis protein with 41 % identity. The LLP12-3 protein predicted a structure of sixα-helix and threeβ-sheets.
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