臺灣博碩士論文加值系統

LLA23為百合花藥 (Lilium longiflorum anther) 成熟晚期表現的花粉專一性基因。LLA23 mRNA轉錄一含142個胺基酸的蛋白質，其C端有一bipartite NLS (nuclear localization signal) 序列。此核定位訊息序列A區有四個離胺酸，B區有二個離胺酸，中間相隔了7個其它不同的胺基酸。為了探討LLA23蛋白質核定位訊息序列的功能，取其C半端的LLA23 cDNA序列接上綠色螢光蛋白質的報導基因，利用竹嵌紋病毒 (Bamboo mosaic potexvirus) 載體在白藜 (Chenopodium quinoa) 葉面上表現外源蛋白質。在感染八天的白藜葉片上可以看到明顯的病斑。利用32P-dCTP標定的綠色螢光蛋白質基因當探針進行北方墨點分析，可在白藜葉內偵測到病毒的重組基因體和兩個重組次基因體RNA。以抗綠色螢光蛋白質的抗體進行西方墨點法檢測，可在白藜葉內檢測出綠色螢光蛋白質。在螢光顯微鏡和雷射共軛焦掃描顯微鏡觀察下，白藜葉片病斑周邊的細胞有聚集成點狀的綠色螢光。DAPI (4'-6-diamidino-2-phenylindole) 染色的結果顯示，點狀的綠色螢光就是聚集在細胞核，而去除了核定位訊息序列的GFP-ΔLLA23融合蛋白質的螢光則散佈於細胞質中，證明LLA23的核定位訊息序列可以把綠色螢光蛋白質帶進核內。將LLA 23蛋白質的核定位訊息序列 A區和B區作定點突變，使離胺酸突變成丙胺酸。突變掉A區四個離胺酸的融合蛋白質進核效率為23 %，而突變掉B區二個離胺酸為57 %，顯示出核定位訊息序列 A區把螢光蛋白質帶進核的效率比B區大。核定位訊息序列 A區四個離胺基若分別突變掉第一、二個離胺酸會顯著的降低融合蛋白質進核的效率，分別為33 %和42 %。假若同時突變掉此二個離胺酸，融合蛋白質的進核效率為31 %，顯示A區的第一、二個離胺酸同等重要，缺一不可。若分別突變A區的第三、四個離胺酸，融合蛋白質進核的效率為幾乎不變。若同時突變掉A區此二個離胺酸，融合蛋白質進核的效率仍為94 %，顯示A區第三、四個離胺酸對融合蛋白質的進核效率沒有影響。總言之，LLA 23蛋白質的核定位訊息序列有入核的功能，其核定位訊息序列 A區的前兩個離胺酸與B區的離胺酸都會影響融合蛋白質入核的功能，而A區的影響顯然較B區重要。本實驗的結果間接証明了LLA 23蛋白質可以進入細胞核。

LLA (Lilium longiflorum anther) 23 expressed at the stage of pollen maturation is a pollen-specific gene. The LLA23 mRNA encodes a protein containing 142 amino acids in which the bipartite nuclear localization signal (NLS) is located at the C-terminus of the polypeptide. The NLS sequence of LLA23 is composed of two regions rich in lysine residues. The A region of NLS contains four lysine residues and the B region of NLS two lysine residues. A segment of seven amino acids other than lysine resides between the two regions of NLS. To study the functional NLS of LLA23 protein, the C-terminal half of LLA23 cDNA sequence was fused with the coding region of a green fluorescence protein (GFP) as a reporter and constructed into bamboo mosaic potexvirus (BaMV) to facilitate infection on leaves of Chenopodium quinoa. After 8 days of infection by BaMV, local lesions on the leaf were clearly observed. Northern blots analyses demonstrated that a recombinant genomic and two recombinant subgenomic RNAs were detected when 32P-dCTP labeled GFP coding region was used as a probe. Immunoblots of leaf protein confirmed that the GFP was detected using anti-GFP antiserum. Under either fluorescence microscope or laser confocal scanning microscopy, the green fluorescence were condensed into spots co-localized with nuclei of the mesophell cells as demonstrated by DAPI (4′-6-diamidino-2-phenylindole) staining. A deletion of the NLS from GFP-ΔLLA23 resulted in the distribution of the green fluorescence in the cytoplasm. It strongly suggested that the NLS of LLA23 has a function of importing GFP into nucleus. Mutagenesis was done either on A or B regions of the NLS by substitution of lysine residue into alanine. Mutagenesis of four lysines together in the A region resulted in 23 % efficiency of locating the fusion protein in the nucleus whereas mutagenesis of the two lysines together in the B region resulted in 57 % importing efficiency. It indicated that A region has more profound influence on nuclear importing than B region. Mutations in the first two lysines in the A region either together or separately reduced to 31-42 % of importing efficiency while no significance of nuclear import was observed when the third and the fourth lysines of A region were mutated either together or separately. In all, the LLA23 NLS has a function of nuclear importing. Both the first two lysine residues at A region and the two lysine residues at B region have the influence on nuclear location. However, the A region has more significant efficiency of importing than that of B region. The data herein demonstrate that LLA23 protein can import into nucleus of the cell.

壹、 中文摘要----------------------------------------------1
貳、 英文摘要----------------------------------------------3
參、 文獻整理----------------------------------------------5
一、 百合--------------------------------------------------5
二、 花藥之發育--------------------------------------------5
三、 與乾燥有關之蛋白和基因之研究--------------------------8
四、 蛋白質進入細胞核機制之相關研究-----------------------11
肆、 材料與方法-------------------------------------------18
一、 GFP-LLA23和GFP-∆LLA23兩融合蛋白質基因之構築----------18
二、 各組融合蛋白質DNA構築體的選殖------------------------19
三、 GFP-LLA23之生體外點突變------------------------------22
四、 各組重組竹嵌紋病毒載體的構築-------------------------23
五、 各組重組竹嵌紋病毒RNA的製備與接種--------------------23
六、 北方墨點法-------------------------------------------24
七、 西方墨點法-------------------------------------------27
八、 顯微鏡觀察-------------------------------------------30
九、 雷射共軛焦掃描顯微鏡數位照片之數據分析---------------30
伍、結果------------------------------------------------------31
一、 GFP-LLA23和GFP-∆LLA23融合蛋白質之構築----------------31
二、 LLA23蛋白質的核定位訊息序列為進核的訊息序列----------31
三、 LLA23蛋白質核定位訊息序列 A與B區的定點突變-----------33
四、 GFP-LLA23蛋白質核定位訊息序列 A區的定點突變----------34
五、 雷射共軛焦掃描顯微鏡觀察結果的螢光量化---------------34
陸、討論------------------------------------------------------37
柒、參考文獻--------------------------------------------------43
捌、圖表------------------------------------------------------55
玖、附圖------------------------------------------------------74

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