I. 鐵炮百合花藥專一基因之表現、定位和功能探討. II. 擬南芥阿拉伯半乳聚醣蛋白AGP31之功能性分析

I. ?用抑制扣除雜合法(suppression subtractive hybridization) 從鐵炮百合(Lilium longiflorum) 花藥的小孢子發育期cDNA 集合庫選殖出幾個花藥專一性基因LLA89、LLA142和LLA66。利用5′-與3′-RACE-PCR延伸而得到LLA89、LLA142和LLA66 cDNA全長序列。LLA89 cDNA 含有一段303 bp 可編譯框架(open reading frame)，可轉譯出100 個胺基酸的酸性蛋白質，其分子量為10.2 kDa。LLA142 cDNA 含有一段171 bp可編譯框架可轉譯出56 個胺基酸的鹼性蛋白質，其分子量為5.7 kDa。LLA66 cDNA可轉譯出308個胺基酸的酸性蛋白質，其分子量為35.7 kDa。LLA89 蛋白質N 端有一段疏水性的訊息胜?(signal peptide) 序列，而LLA142和LLA66則無。序列比對後顯示LLA89 即是已知的百合LIM4; LLA142是嶄新的未知蛋白質，而LLA66蛋白質與許多物種之cis-prenyltransferase有高達30-41%相同度，然而和單子葉植物cis-prenyltransferase之演化不同。根據決定已知cis-prenyltransferase長度的關鍵標記的胺基酸，LLA66被認為可合成長鏈聚戊烯產物 (long-chain polyprenyl products)。利用北方墨漬法分析，得知LLA89 、LLA142 和LLA66具花藥專一性。利用digoxigenin-labeled riboprobe進行原位雜合(in situ hybridization) 的實驗證實三者的mRNA在花藥壁的絨氈層中呈現強烈的訊號。LLA89 基因會受外加的激勃素(gibberellin) 誘導而表現，LLA142和LLA66 則不會，然而三者皆會受內生性激勃素的誘導而產生。利用激勃素抑制劑uniconazol和乙烯抑制劑2,5-norbornaddien處理，顯示LLA66基因受內生的激勃素誘導而表現，但是不受乙烯的調控。利用TAIL-PCR找到LLA66基因啟動子調控區域。在小孢子生長時期，花藥中prenyltransferase的活性與絨氈層的生長與分解是互相協同的。將LLA66可編譯框架構築到表現載體pYES2/CT並轉殖到酵母菌Saccharomyces cerevisiae表達外源蛋白，再使用Ni2+–nitrilotriacetic acid–agarose純化。體外(in vitro)酵素活性分析顯示LLA66可催化合成聚戊烯基雙磷酸酯 (polyprenyl diphosphates)。酵素反應最適的Mg2+濃度為0.2 mM；反應最適的pH值和溫度分別為7.0和50°C。酵素和受質(substrate)之親和性為km= 5.7 ?M。我們推測百合花藥絨氈層cis-prenyltransferase可能參與dolichols和polyprenols compounds的合成，以協助小孢子壁的形成。II. 此研究工作主要探討花粉管在雌蕊中生長過程中，阿拉伯半乳聚醣蛋白(AGP31)基因的功能。我們建構RNAi載體並成功轉殖到野生型(WT)阿拉伯芥中。RNAi突變株聚呈現較小且深綠色的表現型，並且果莢短小。正反交試驗(reciprocal cross)中，當利用野生型之花粉授粉RNAi突變株之雌蕊，其果莢短小；而相反的利用RNAi突變株之花粉授粉野生型之雌蕊，其果莢長度與控制組相當，此結果證實RNAi突變株造成雌蕊缺陷而導致果莢短小以及種子數目較少。透過Blue Dot assay發現在RNAi突變株雌蕊中，花粉管進入胚株的數目較少於在野生型中。此外，利用苯胺藍(aniline blue)染色已授粉之雌蕊結果發現，在RNAi突變株雌蕊中，花粉管進入雌蕊的數量較少且生長速度也較慢。這些結果顯示RNAi突變體會影響花粉管在雌蕊中的生長，推測AGP31蛋白在雌蕊傳輸組織(transmitting tissue)中扮演控制花粉生長的角色。BiFC assay證實AGP31蛋白自己可結合形成二聚體(dimer)且表現在細胞壁。分別利用BiFC assay和pull-down assay證實AGP31蛋白可與花粉管專一表現的receptor-like kinase (RLK) 5 和ANXUR2 (RLK12)相互結合。綜合以上之結果，在雌蕊傳輸組織表現的AGP31蛋白，透過與花粉管之接收器(receptor-like kinase)相互結合來控制花粉管的生長。I. A number of stage-specific genes have been isolated from a suppression subtractive cDNA library constructed from developing anthers of L. longiflorum. 5′- and 3′-RACE-PCR were used to obtain the full length cDNA sequences. The LLA89 cDNA encodes an acidic polypeptide of 100 amino acids with a calculated molecular mass of 10.2 kDa. The LLA142 cDNA encodes a basic polypeptide of 56 amino acids with a calculated molecular mass of 5.7 kDa. The LLA66 cDNA encodes a polypeptide of 308 amino acids with a calculated molecular mass of 35.7 kDa. The LLA89 protein had a strong hydrophobic region at the N-terminus, indicating the presence of a signal peptide whereas the LLA142 and LLA66 do not have. Sequence alignment revealed that the LLA89 protein is identical to a reported LIM4 (Lily messages Induced at Meiosis) protein with unknown function, while the LLA142 protein is a novel protein. Sequence alignment revealed that the LLA66 protein shares 30-41% identity with cis-prenyltransferases of various broad-spectrum species and is phylogenetically distinct from other monocot cis-prenyltransferases. Based on critical regulatory domains in cis-prenyltransferase, LLA66 was concluded to catalyze the production of long-chain polyprenyl products. RNA blot analysis indicated that the transcripts of LLA89, LLA142 and LLA66 were anther-specific and differentially detected in the anther wall and in the microspore of developing anthers. In situ hybridization with digoxigenin-labeled riboprobes for the three genes revealed strong signals localized to the tapetal layer of the anther wall. The LLA89 gene could be exogenously induced by gibberellin (GA) while the LLA142 and LLA66 genes could not. However, these three genes are induced by endogenous GA. Furthermore, GA inhibitor analysis indicated that the LLA66 gene is endogenously induced by GA, but its induction is independent of ethylene regulation. Thermal asymmetric interlaced (TAIL)-PCR was employed to obtain the 5'-regulatory region of LLA66. The cis-prenyltransferase gene, LLA66, was the first prenyltransferase to be identified in the tapetum and microspores. The enzyme activity of prenyltransferases in various stages of microspore development correlated with tapetal growth and disintegration. The coding region of LLA66 constructed in pYES2/CT vector was introduced into Saccharomyces cerevisiae, and the His-tagged LLA66 protein was affinity-purified using Ni2+-nitrilotriacetic acid-agarose. In vitro enzymatic assay revealed that the enzyme catalyzed the formation of polyprenyl diphosphates. A drastic increase of enzyme activity was detected with the addition of Mg2+ ion up to 0.2 mM. A further addition of Mg2 + ion inhibited the enzyme activity. The optimum pH value and temperature of the enzyme were found to be pH 7.0 and 50 °C, respectively. The enzyme exhibited an affinity for isopentenyl diphosphate (IPP), with a Km value of 5.7 μM. The involvement of cis-prenyltransferase in the anther in the synthesis of dolichols and polyprenols is discussed. II. For this work, we mainly investigated the function of arabinogalactan protein AGP31 gene from Arabidopsis during pollen tubes growth in the transmitting tissue. Using RNAi-mediated gene silencing strategy to generate AGP31 knock-down mutants. The phenotype of RNAi mutants with AGP31 transcript reduced to almost knock-out level was small and dark green and the length of siliques in RNAi mutant was shorter than in wild type (WT) plants. Reciprocal crossed with WT using a severe RNAi knock-down line as a female parent resulted in shorter siliques and lower seeds relative to WT, but not when used as the male parent. These results indicates that RNAi caused the defect in female part. Visualization of pollen growth in the pistil by 'blue dot assay' after limited pollination by WT pollen showed that fewer pollen tubes reached the ovules in the pistils of the RNAi line compared with WT. In addition, time series of pollen tube growth stained with aniline blue showed fewer pollen tubes and their growth was slower in the pistil of the RNAi mutant compared with WT. Taken together, these results are consistent with AGP31 protein playing an important role in regulating pollen tube growth. Bimolecular fluorescence complementation (BiFC) assays showed AGP31 can interact by itself and showed AGP31 is cell wall localization. Furthermore, using BiFC and pull-down assays, we also showed that AGP31 interacts with two pollen-specific FERONIA-related receptor-like kinases (RLKs), RLK5 and ANXUR2 (RLK12). The transmitting tissue of pistils provides the passage for pollen tube growth, AGP31 may be a guider during pollen tube growing through transmitting tissue.RESEARCH PURPOSE.....................................................................................................................................2 Chapter I. Expression, localization and function of anther-specific genes in Lilium longiflorum ABSTRACT(ENGLISH).....................................................................................................................................3 ABSTRACT(CHINESE)…………………………………………………..............................................................5 INTRODUCTION…….....................…......………….........................................................................................6 MATERIALS AND METHODS….....…...........................................................................................................15 RESULTS.........................................................................................................................................................22 DISCUSSION.….....……..................................................................................................................................31 REFERENCES………………..................................................................................….…………….……….….36 TABLES AND FIGURES…………...................................................................................................................49 Chapter II. Functional analysis of Arabidopsis arabinogalactan protein AGP 31 in pollen tube growth ABSTRACT(ENGLISH)....................................................................................................................................78 ABSTRACT(CHINESE)………………………………………………….............................................................79 INTRODUCTION…….....................…......…………........................................................................................80 MATERIALS AND METHODS….....…............................................................................................................83 RESULTS..........................................................................................................................................................86 DISCUSSION.….....……...................................................................................................................................89 REFERENCES………………..................................................................................….……………..……….….91 TABLES AND FIGURES…………....................................................................................................................9