文心蘭花朵發育相關之MADS box 基因之選殖及功能分析

Abstract

文心蘭為重要的單子葉觀賞花卉，是切花市場上極具經濟價值的花卉，但是與文心蘭花朵發育的研究報告卻極少，因此本研究的目的在選殖及分析與文心蘭花朵發育相關之MADS box基因，並對這些基因的功能作深入的分析。 首先由文心蘭中選殖到一個MADS box基因，OMADS3 (Oncidium Gower Ramsey MADS box gene 3)。OMADS3高度相似於B功能群MADS box基因中的paleoAP3演化分支 (paleoAP3 lineage) 及TM6演化分支 (TM6 lineage)，但OMADS3缺乏B群MADS box基因C端的保守序列 (consensus motif)。有別於B群MADS box基因，OMADS3 mRNA在花器及營養葉中皆有表現，且在文心蘭的基因組中，可能存在二或三種不同的染色體位置結構 (position organization)。35S::OMADS3擬南芥轉殖株在花序頂端形成終結花序 (terminal flower) 的現象，類似A群MADS box基因AP1之35S::AP1轉基因擬南芥。而35S::OMADS3ΔC及35S::OMADS3ΔM轉植株，又出現類似A群MADS box基因突變種ap2的性狀，如花萼轉形成雌蕊狀結構，花瓣轉形成雄蕊狀結構。此結果顯示OMADS3可能為單子葉植物中序列為較原始形式的TM6基因，而功能卻如同A群MADS box基因，調控花器形成及開花啟始的過程(第二章)。 在大量表現文心蘭屬於AP1/AGL9基因群之OMADS1的轉殖擬南芥植株出現捲曲葉、極度早開花、終結花序、及花萼轉形成雌蕊狀結構，花瓣則轉形成雄蕊狀之結構。35S::OMADS1轉殖菸草亦出現提早開花的性狀。在35S::OMADS1轉殖擬南芥植株中，參與開花誘導與起始的基因FT、SOC1、LEAFY 和AP1明顯被誘導表現。35S::OMADS1轉殖至晚開花突變株中可救回gi、co而對於ft及fwa則無效。因此FT及SOC1可能是OMADS1在擬南芥中的目標基因，透過活化這兩個基因進而促進開花。此外亦探討擬南芥AGL6是否有類似OMADS1的作用。結果發現35S::AGL6-sense與35S::OMADS1轉殖擬南芥一樣，有極早開花的性狀。35S::AGL6-antisense轉殖株則與一般野生型無異 (第三章)。 另外自文心蘭年輕花苞中選殖到1個 D功能的MADS box基因， OMADS2其只在雌蕊中表現，及1個C功能的MADS box基因，OMADS4其在雄蕊與雌蕊表現。OMADS2及OMADS4在文心蘭的基因組中，可能存在二或三種不同的染色體位置結構 (position organization)。35S::OMADS2與35S::OMADS4轉殖株外觀與一般野生型無異，但開花時間比野生型稍早(第四章)。 進一步以Yeast Two-hybrid分析的結果顯示OMADS3及OMADS1自身皆可形成homodimers，但彼此亦可形成heterodimers。OMADS4不會形成homodimers，但與OMADS1具有相當的結合力。OMADS2則與其他三個蛋白質皆沒有作用。藉由C terminal truncation對OMADS3的C端進行不同長度的截切，比較其形成homodimers及與OMADS1形成heterodimers的能力變化。OMADS3形成homodimers的能力在保留完整C端時為最高，需保留40個胺基酸，才有可能形成homodimers，而第40個到第50個胺基酸是關鍵區域。至於OMADS1與OMADS3形成heterodimers的能力，只要保有OMADS3 C端40個胺基酸，兩者的作用力都能維持一定強度；OMADS3 C端第30個到第40個胺基酸，是與OMADS3形成heterodimers的必要區域。本結果佐證文心蘭中的MADS box蛋白質也是以多重聚合物 (multimeric) 的形式共同作用，以達成花器形成與開花啟始(第五章)。 為擴展文心蘭花朵發育相關之MADS box 基因的族群與研究，本研究並建立了文心蘭年輕花苞cDNA基因庫，其重組率為91%，平均插入片段大小為0.9 kb。此基因庫對建立文心蘭花苞基因表現模式而言是一項很好的資源，亦可提供建立cDNA基因庫微陣列 (microarray)，用以大量篩選文心蘭MADS box基因(第六章)。

Although orchids are among the most important plants in the flower market around the world, little research on MADS box genes has been reported. The nearly identical shape of sepals and petals as well as the production of a unique large lip in orchid flower make it as a very special plant species in the study of flower development. Therefore, the isolation of MADS box genes and further study of their roles on an orchid (Oncidium Gower Ramsey) flower development is the goal for this study. OMADS3, isolated and characterized from Oncidium Gower Ramsey. OMADS3, showed high sequence homology to both paleoAP3 and TM6 lineage of B group MADS box gene. Despite the sequence homology, consensus motifs identified in the C-terminal region of B group genes were absent in OMADS3. Southern analysis indicated that OMADS3 was present in O. Gower Ramsey genome in low copy numbers. Different from most B group genes, OMADS3 mRNA was detected in all four floral organs as well as in vegetative leaves. 35S::OMADS3 transgenic plants showed novel phenotypes by producing terminal flowers similar to those observed in transgenic plants ectopically expressed A functional genes such as AP1. Ectopic expression of OMADS3 cDNA truncated with the MADS box or C terminal region in Arabidopsis generated novel ap2-like flowers in which sepals and petals were converted into carpel-like and stamen-like structures. Our results suggested that OMADS3 might represent an ancestral form of TM6-like gene which was conserved in monocots with a function similar to A functional gene in regulating flower formation as well as floral initiation (Chapter 2). An AP1/AGL9 group of MADS box gene, OMADS1, with extensive homology to the Arabidopsis AGAMOUS-like 6 gene (AGL6) was characterized from orchid (Oncidium Gower Ramsey). Transgenic Arabidopsis and tobacco ectopically expressed OMADS1 showed similar novel phenotypes by significantly reducing plant size, flowering extremely early, and losing inflorescence indeterminacy. In addition, homeotic conversion of sepals into carpel-like structures and petals into staminoid structures were also observed in flowers of 35S::OMADS1 Arabidopsis. This result indicated that OMADS1 was involved in floral formation and initiation in transgenic plants. Further analysis indicated that the expression of flowering time genes FT, SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) and flower meristem identity genes LEAFY (LFY), APETALA1 (AP1) was significantly up-regulated in 35S::OMADS1 transgenic Arabidopsis plants. Furthermore, ectopic expression of OMADS1 rescued late-flowering phenotype in gi-1, co-3 but not for ft-1 and fwa-1 mutants. These results supported that ectopic expression of OMADS1 influenced flower transition and formation by acting as an activator for FT and SOC1 in Arabidopsis (Chapter 3). In addition, OMADS2 showing high homology to the D functional MADS box gene and OMADS4 showing high homology to the C functional MADS box gene were isolated and characterized in orchid (Oncidium Gower Ramsey). The expression of OMADS2 mRNA was restricted to the carpel and was absent in the other flower organs or vegetative leaves. OMADS4 mRNA was detected in stamen and carpel. Both OMADS2 and OMADS4 were present in O. Gower Ramsey genome in low copy numbers. Ectopically expressed OMADS2 or OMADS4 flowered lightly earlier than wild-type plants (Chapter 4). Yeast two-hybrid analysis indicated that OMADS3 is able to strongly interact with OMADS1. Both proteins are also able to form homodimers. OMADS4 is able to interact with OMADS1. By contrast, OMADS2 is not able to interact with OMADS1, 3 or 4. At least 40 amino acids left in the C-terminal region of OMADS3 is essential for homodimer formation of OMADS3. However, 30-40 amino acids left in the C-terminal region of OMADS3 is necessary for interaction between OMADS3 and OMADS1. These results strongly indicated that MADS proteins in orchid are also functioning as multimers (Chapter 5). Finally, a cDNA library made from developing flower of Oncidium Gower Ramsey was constructed. 91% of the clones in this library contained an insertion with an average size of 0.9 kb. This library can be used to screen more flower specific MADS box genes in the future (Chapter 6).