文心蘭黃色素生合成相關基因之選殖與分析

Abstract

為創新蝴蝶蘭的花色，本論文進行文心蘭黃色素生合成相關基因之選殖與分析，以期將來轉殖至蝴蝶蘭。以次選殖得到之金魚草aureusidin合成酶基因AmAS1 488 bp片段為探針，篩選文心蘭基因組庫，選取選殖系λOgAS9進行限制酶圖譜及南方氏雜交分析，接著進行次選殖及定序，取得文心蘭之aureusidin synthase基因，命名為OgAS1 (GenBank accession number AY858697)，此基因長2,145個鹼基對，具有2個顯子及1個長507 bp的隱子，可解碼出545個胺基酸，預測之分子量為62.7 KDa。OgAS1解碼之胺基酸序列與鳳梨之多酚氧化酶 (polyphenol oxidase) 最相似，相似度達44.4%。進行南方氏及北方雜交分析得知，OgAS1非單一拷貝基因且主要在花器中進行表達。為了瞭解OgAS1之功能，將OgAS1解碼區連接於2 X CaMV 35S啟動子下游，構築得到基因表達質體，利用基因槍轉殖法進行金魚草、蝴蝶蘭及文心蘭花瓣之轉殖，並運用HPLC，分析轉殖前後花瓣內色素的組成及含量變化。未轉殖黃花金魚草花瓣萃取物之HPLC分析，在滯留時間12.57分鐘時可見一明顯波峰為aureusidin，其最大吸收波峰在405 nm。黃花金魚草花瓣單轉殖文心蘭OgAS1基因及金魚草AmAS1基因之試驗組皆可使aureusidin波峰顯著升高，白花金魚草經轉殖金魚草或文心蘭aureusidin synthase基因後，aureusidin波峰皆有下降趨勢。另白花蝴蝶蘭花瓣共轉殖蝴蝶蘭苯基苯乙烯酮合成酶 (chalcone synthase, CHS) 及金魚草AmAS1基因後，於405 nm下有一滯留時間為12.7分鐘之物質有上升的趨勢，其最大吸收波峰為370 nm，推測為chalcone之ㄧ種，待進一步分析以釐清基因功能及產物種類。轉殖文心蘭OgAS1基因則使波峰明顯下降，推測可能是因為轉入同源基因造成基因默化現象。在啟動子序列分析部份，由與資料庫比對結果得知OgAS1啟動子上游具有多個調控保守性序列，包括auxin荷爾蒙、光線、創傷、抗病機制等。

In order to create new color of Phalaenopsis flower, a yellow pigment biosynthesis-related gene, aureusidin synthase gene, was cloned and analyzed from Oncidium. The gene fragment of aureusidin synthase from snapdragon (AmAS1) was used as probe to screen a genomic library of Oncidium. Based on restriction endonuclease maps and sequence analysis of genomic clone λOgAS9, aureusidin synthase gene from Oncidium was obtained and named OgAS1 (accession number AY858697 in GenBank). Nucleotide sequence analysis revealed that OgAS1 is 2,145 bp in length and contains two exons and one intron of 507 bp. OgAS1 encodes a polypeptide of 545 amino acid residues whose predicted molecular weight is 62.7 KDa. The deduced amino acid sequences of OgAS1 showed the highest homology with the duduced amino acid sequences of polyphenol oxidase gene from pineapple for 44.4％. Based on the result of Southern blot analysis, aureusidin synthase gene from Oncidium belongs to high-copy gene. The accumulation of mRNA was found primarily in the flowers. To understand the function of OgAS1, the open reading frame of OgAS1 was constructed into gene expression vector driven by 2 X CaMV 35S promoter. The HPLC chromatographic profile of the extract of untransformed yellow snapdragon revealed that the obvious peak at retention time 12.57 min was aureusidin which had the maximum absorption wavelength at 405 nm. Transient expression analysis of OgAS1 and AmAS1 in yellow snapdragon petals by particle bombardment displayed that the peak of aureusidin rose. However, transient expression analysis of OgAS1 and AmAS1 in white snapdragon petals showed the peak was downward. Co-transformation of chalcone synthase (CHS) gene from Phalaenopsis and AmAS1 in Phalaenopsis petals resulted that a rising peak of an unknown chemical whose retention time was 12.7 min and its maximum absorption wavelength was 370 nm. That was presumed to be a kind of chalcone. The HPLC chromatographic profile for yellow Oncidium petals transformed with OgAS1 revealed the peak of aureusidin was downward obviously. Gene silencing caused by transformed homologous gene could be one possible explanation for this phenomenon. In the promoter sequence analysis of Oncidium aureusidin synthase gene, many predicted responsive elements related to auxin, light, wounding and disease resistance were found.