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|標題:||百合花粉與乾燥有關 LLA-32 cDNA 之篩選與聚半乳醣醛酸酵素 cDNA 之選殖
Cloning and Identification of a LLA-32 cDNA Related to Desiccation and a cDNA Encoding Polygalacturonase during Development in Lilium longiflorum
|關鍵字:||pollen desiccation;花粉乾燥;N-terminal sequence;degenerate primer;polygalacturonase;allergen;LLA-32 protein;氨基酸定序;退化引子;聚半乳醛酸酵素;過敏原;LLA-32 蛋白質||出版社:||農業生物科技學研究所||摘要:||
植物花粉的發育是一連串複雜分化的過程。在百合花藥中，小孢子母細胞經過減數分裂進入四分子期，產生小孢子。每個小孢子再經過一次有絲分裂而形成兩個細胞的成熟花粉。在花粉成熟的最後一個階段必須經過不等程度的乾燥失水過程。百合花粉乾燥的過程中會有一些蛋白質累積表現，其中一個蛋白質 (LLA-32) 的分子量為32 kDa。為了選殖 LLA-32 蛋白質的cDNA，我們先將LLA-32蛋白質的片段分別作氨基酸定序後，利用這些氨基酸序列設計退化引子，並以RT-PCR 的技術取得LLA-32 cDNA (LLP-B3) 。此cDNA 片段長度有343 bp。北方墨點分析顯示此LLP-B3片段所雜合的 mRNA 具有量多、花粉專一和花粉成熟期才表現的特性。此與已知的LLA-32蛋白質的特性相符。經比對更發現此片段與蕃茄花粉中與乾燥有關的LAT 52 蛋白質及數個花粉過敏原有極高的相同度。此外，在整個篩選的過程中也得到了一長度約為500 bp 的LLP-A1 cDNA 片段。經由北方墨點分析顯示LLP-A1 mRNA 同樣具有量多、花粉專一和花粉成熟期表現的特性。因此，以此片段為探針，對百合成熟花粉的 cDNA 集合庫進行篩選，得到了一完整的cDNA。它包含了5’ 端非轉譯區的61 bp，轉譯區的891 bp及3’ 端非轉譯區的218 bp。此完整的cDNA 可轉譯296個氨基酸。經由比對後發現其與數個聚半乳醣醛酸酵素有極高的相同度。從蛋白質結構分析，此百合聚半乳醣醛酸酵素為一親水性蛋白質，於N端約10個氨基酸的區域有所謂的signal peptide。百合聚半乳醣醛酸酵素是屬於C 類的聚半乳醣醛酸酵素。
Pollen development is a series of complex differentiation. Within the lily anthers, pollen mother cells undergo meiosis to produce microspores at the tetrad stages. Consequently, each microspore goes on the first run of mitosis, resulting in the formation of bicellular and mature pollen. At the final stage of maturation, pollen exhibits various degrees of desiccation. During pollen development a subset of protein including the LLA-32 proteins accumulate upon desiccation. To screen and identify the clones encoding LLA-32 proteins in lily pollen, the purified LLA-32 protein was subjected to Lys-C digestion, producing five peptide fragments. The five peptides were sequenced and degenerate primers were designated from the suitable sequences resulted from peptide sequencing. By the use of RT-PCR, a piece of LLA-32 cDNA fragment (LLP-B3) was obtained and sequenced. The LLP-B3 cDNA is 343 bp long. Northern hybridization revealed that the LLP-B3 mRNA is abundant、organ-specific and expressed at the maturation stage of pollen development. Sequence analysis exhibits significant similarity between the amino acid sequence predicted from the LLP-B3 cDNA and a tomato LAT52 which is related to desiccation. The sequence of LLP-B3 is also similar to a group of allergenic proteins. Besides, a fragment of LLP-A1 cDNA was obtained during the screening process. Northern hybridization revealed that the LLP-A1 mRNA is abundant, organ-specific and expressed at the stage of pollen maturation, which are also characteristic of LLA-32 gene products. Therefore, the LLP-A1 cDNA was used as a probe to screen a cDNA library of lily pollen and a full-length cDNA was obtained. The cDNA has 1167 bp in length, containing 61 bp at the 5’-untranslated region, an 891 bp at the coding region encoding 296 amino acids, and 218 bp at the 3’-untranslated region. Sequence analysis exhibits significant similarity of the lily PG protein to a group of polygalacturonases of various species. The lily PG is a hydrophilic protein that contains a signal peptide at the N-terminus. It is a member of clade C polygalacturonase.
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