文心蘭B群MADS box基因之功能分析與應用

Abstract

為了瞭解南西文心蘭花器的發育機制，從已知的文心蘭MADS基因中選殖具花被特異表達的B群功能性MADS box 基因，OMADS5及OMADS9進行研究。此二個基因屬paleoAP3的分支，其基因表達量OMADS5 mRNA不在唇瓣表現外，在三唇瓣突變株中的表現量也有明顯的下降；OMADS9 mRNA除了表現在花瓣及唇瓣部位，三唇瓣中唇瓣化的部位也有很高的表現量。由此認為OMADS5在唇瓣的形成可能扮演負調控的角色。為了直接瞭解在文心蘭中OMADS5對唇瓣發育的關係，在本篇研究中我們從文心蘭基因體DNA中藉由基因體步行的方式選殖出約2.3 kb的OMADS5啟動子。此外我們將含有35S::OMADS5的pEpyon-32H藉由農桿菌EHA105轉入45天大的文心蘭擬圓球體(PLBs)中，再將受感染的PLBs放置在含有8 μg/ml hygromycin的G10培養基中篩選。接著萃取非轉殖株及轉殖株PLB的全部DNA，以抗性基因(hptII)和OMADS5基因進行PCR來鑑定轉基因植物。為增加轉基因的成功率，利用不同品系文心蘭PLB進行轉基因測試，並以效率佳者作為35S::OMADS5及35S::OMADS9基因轉殖的材料。為了藉由農桿菌基因轉殖而以基因靜默的方式探討OMADS5及OMADS9的功能並且針對文心蘭花形進行改造，故構築出OMADS5 RANi、OMADS9 RNAi以及OMADS5和OMADS9接上SRDX、VP16蛋白融合序列。我們也從文心蘭基因體中選殖到一段含內含子(intron)的α-tubulin片段，並以此內含子構築出文心蘭RNAi轉接載體。由於文心蘭的新品種難以產生，所以期望利用分子育種的技術來創造出新的花器型態，因此藉由調控B功能性MADS box基因預期可能使花被發生型態上的改變。未來文心蘭除了可以改變其花色和花的大小之外，還可以創造出具有各式各樣不同花形的文心蘭。

To investigate floral formation in Oncidium ‘Gower Ramsey', two paleoAPETALA3 (paleoAP3) genes, O. Grower Ramsey MADS box gene5 (OMADS5) and OMADS9 were characterized in our laboratory. The OMADS5 mRNA was absent in lips and was significantly down-regulated in lip-like organs in the peloric mutant flowers. In addition, the mRNA for OMADS9 was expressed in lip and petals and a high amount of OMADS9 expression was also observed in lip-like organs of peloric mutant flowers. This suggested a possible negative role for OMADS5 in regulating lip formation. To explore the function of OMADS5 in lip formation, we first cloned OMADS5 promoter with 2.3 kb by genomic walking. In addition, the vector pEpyon-32H containing OMADS5 was transformed into 45-day-old Oncidium protocorm-like body (PLB) via Agrobacterium tumefaciens. The transformants were selected by 8 μg/ml hygromycin G10 medium. To confirm that these plants were transformants, total DNA was isolated and analyzed by PCR from PLBs of four transgenic plants to find the appearance of the antibiotic resistant gene (hptII) and OMADS5 gene. We further used new PLB with high efficiency of transformation to perform the 35S::OMADS5 and 35S::OMADS9 transformation. Furthermore, to modify the floral shape of Oncidium, OMADS5 RNAi and OMADS9 RNAi as well as OMADS5/OMADS9 fused with either SRDX or VP16 sequence were constructed and transformed into Oncidium. In order to obtain a useful RNAi transferred construct in Oncidium, an α-tubulin intron from Oncidium genomic DNA was cloned. New Oncidium varieties are difficult to generate; hence molecular breeding is an attractive approach to create a new flower shape. It is expected that the manipulation of B function MADS box genes in this study will potentially convert perianths into differential shapes.