鐵炮百合花藥專一表現之嶄新類黏附蛋白基因LLA1271 之調控和功能

LLA1271 為絨氈層和小孢子專一表現的基因，編譯出 24 kDa 的蛋白質，其 N 端 26 個胺基酸為訊號胜肽，並含有八個高度保留的重複序列。序列比對分析顯示 LLA1271 與冬蟲夏草 (Ophiocordyceps sinensis) 的GLEYA adhesin domain protein 具有 37%的相同度。黏附蛋白 (adhesin) 是真菌或細菌表面上特有的蛋白質，百合 LLA1271 為首次在高等植物中發現到的類黏附蛋白 (adhesin-like protein) 。我們將 LLA1271 基因構築在絨氈層專一的 TAP 啟動子上，以農桿菌感染轉型至阿拉伯芥 (Arabidopsisthaliana) 中，篩選出三株 T2-homologous 子代植株。野生型花粉萌發率將近 90%，而 TAP::LLA1271 轉殖株花粉萌發率下降至 70%，且TAP::LLA1271 轉殖株果夾也較野生型短小。透過掃描式電子顯微鏡觀察花粉外觀，與野生型相比，TAP::LLA1271 轉殖株花粉外壁網狀結構有不正常的堆積，改變了外壁的形成。LLA1271 為一獨特的類黏附蛋白可由絨氈層分泌到小孢子表面且會影響花粉外壁結構的形成。為了找到LLA1271 的啟動子，利用熱不對稱交錯聚合酶連鎖反應 (thermalasymmetric interlaced PCR) 向 5'UTR 延伸。第一次延伸得到 199 bp，再次延伸得到 854 bp，總共延伸 1053 bp。將序列經過 PLACE database 進行cis-element 分析，結果顯示 LLA1271 啟動子具有 10 個可能的花粉專一的調控區，以及數個植物荷爾蒙調控區。建構 LLA1271 啟動子不同長度的5'-去除片段，構築到 β-glucuronidase (GUS)基因，轉入阿拉伯芥中觀察到LLA1271 基因會專一表現在植株的花藥。影響花藥專一表現的啟動子調控決定區位於轉錄起始位置上游-554 和-370 bp 之間，此區域也預測到 2個花藥專一表現調控區 GTGA 及 AGAAA motifs。另外，LLA1271f-1010::barnase 轉殖株呈現雄不稔的現象，果莢異常短小，幾乎不含有種子。切片觀察 LLA1271f-1010::barnase 轉殖株的花藥早在阿拉伯芥第五時期，也就是小孢子母細胞出現的時期，絨氈層及小孢子母細胞就出現異常的液泡化，導致無正常花粉產生。將 LLA1271f-1010::barnase 轉殖株雌配子與野生型阿拉伯芥花粉雜交後，植株果莢正常發育，種子也恢復產出。LLA1271 為一個高等植物新穎的類黏附蛋白，專一表現在絨氈層及小孢子，由絨氈層分泌到小孢子外壁，影響小孢子和花粉外壁的形成。LLA1271 is a tapetum/microspore-specific gene that encodes an adhesin-like protein with a molecular mass of 24 kDa. LLA1271 protein contains a signal peptide of 26 amino acids at the N-terminus and eight highly conserved sequence repeats. Sequence alignment analysis revealed 37% identity between LLA1271 and Os (Ophiocordyceps sinensis) adhesin, a GLEYA adhesin domain protein. Adhesins are specific surface proteins found in bacteria and fungi; the lily LLA1271 is an adhesin- like protein first found in higher plants. The coding region of LLA1271 gene was fused with a rice tapetum-specific gene regulatory region (TAP) and transformed into Arabidopsis plants. Three T2-homologous plants were obtained. The pollen of TAP::LLA1271 lines showed lower germination percentage (about 70%) than that of the wild type (90%). In addition, TAP::LLA1271 siliques displayed shorter length than wild- type siliques. Scanning electron microscopy of the TAP::LLA1271 pollen showed abnormal deposition and mild distortion of exine formation. We proposed that the LLA1271 protein once synthesized in both the tapetum and microspore is secreted and deposited on the surface of microspores affecting exine formation and patterning. Using the TAIL-PCR (thermal asymmetric interlaced PCR) approach, we identified a total fragment of 1053 bp upstream the start codon obtained by the first extension of 199 bp and 854 bp from the second extension of TAIL-PCR. Based on PLACE database, the LLA1271 promoter contains ten potential pollen-specific and several hormone-regulated cis-elements.The various 5'-deleted fragements of LLA1271 promoter were constructed, fused with the GUS reporter gene, and introduced into Arabidopsis for functional analysis. GUS activity was specifically detected in the anthers of transgenic lines with the full-length, and partial promoter fragements of - 802 and -554 bp upstream the putative transcription start site (TSS). However, no signal was detected in the anther when the fragment was deleted down to -369 bp or less upstream the putative TSS, suggesting that the fragement between -554 bp and - 370 bp is a decisive regulatory region required for the anther-specificity, that is also supported by the existence of one GTGA and one AGAAA motifs, the two anther- specific cis-elements. In addition, LLA1271f-1010::barnase transgenic lines exhibited complete male sterility and extremely short and seedless siliques. Cross-sections of LLA1271f-1010::barnase anthers revealed that at stage 5 where the microspore mother cells form in the anther, the microspore mother cells and tapetum vacuolated, resulting in the production of abnormal patthern. When female organ of LLA1271f- 1010::barnase transgenic lines was cross-pollinated with wild-type pollen, the resulting siliques were reverted to a size similar to the wild-type siliques, suggesting that the female organ does not play a role in deformation. LLA1271 is a unique adhesion-like protein in higher plants. The protein is produced both in the tapetum and microspores, secreted and deposited on the surface of microspores affecting exine formation and patterning.中文摘要 ...................3 英文摘要 ...................4 前言 ...................5 壹、前人研究 ...................6 一、花藥的發育與構造 ...................6 二、絨氈層的發育和花粉壁的生成 ...................6 三、影響花藥發育的專一性和調控基因 ...................8 四、花藥專一啟動子與雄不稔 ...................10 五、真菌黏附蛋白...................11 貳、材料與方法...................14 一、總 RNA 萃取/反轉錄 cDNA ...................14 1. 阿拉伯芥總 RNA 萃取...................14 2. 反轉錄聚合酶連鎖反應 (reverse transcription, RT-PCR) ....... 14 3. DNA 電泳...................14 4. 聚合酶鏈鎖產物之純化回收...................15 二、西方墨漬法 (Western blotting) ...................15 1. 總蛋白質萃取...................15 2. 蛋白質濃度測定 ...................15 3. SDS 電泳 (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) ...................16 4. 西方墨點轉漬 ...................16 5. 蛋白質免疫測試 ...................16 6. 抗體的製備與墨點親和 (blot-affinity) 純化...................17 三、百合 LLA1271 啟動子鑑定 ...................17 1. 百合 genomic DNA 萃取 ...................17 2. 熱不對稱性交錯聚合酶連鎖反應 (thermal asymmetric interlaced ploymerase chain reaction, TAIL-PCR) ...................17 3. 南方墨漬法 (Southern blotting) ...................18 4. 雜合探針製備 ...................18 5. 雜合反應 ...................19 6. DNA 黏接反應 (Ligation) ...................19 7. 質體之轉型作用 (Transformation) ...................19 8. 質體 DNA 之萃取 ...................19 四、阿拉伯芥轉殖...................20 1. 農桿菌勝任細胞之製備 ...................20 2. 農桿菌快速冷凍轉型 (freeze-thaw) ...................20 3. 阿拉伯芥之基因轉殖 ...................20 4. 轉基因植物之篩選 ...................20 五、阿拉伯芥轉殖株花粉觀察 ...................21 1. 花粉萌發 ...................21 2. 亞歷山大染色法 (Alexander stain)...................21 3. 掃描式電子顯微鏡...................21 4. 切片處理和穿透式電子顯微鏡...................22 5. GUS 呈色 ...................22 参、結果...................23 一、LLA1271 序列比對分析...................23 二、TAP::LLA1271 轉殖株的鑑定和表現 mRNA 和蛋白質的差異 .. 23 三、TAP::LLA1271 轉殖株造成果莢短小和花粉萌發率下降 ............ 24 四、TAP::1271 轉殖株造成花粉外壁發育不完全 ...................24 五、LLA1271 基因啟動子之鑑定和分析 ...................25 六、阿拉伯芥 LLA1271::GUS 轉殖株顯示 GUS 只表現在花藥絨氈層.........25 七、LLA1271f-1010::barnase 轉殖株造成嚴重的雄不稔表型 .........26 肆、討論 ...................27 伍、參考文獻 ...................31 陸、圖表...................38 柒、附圖...................5