蝴蝶蘭苯基苯乙烯酮合成酶cDNA之選殖及分析

Molecular Cloning and Analysis of the cDNA Clones Encoding Chalcone Synthase in Phalaenopsis

蝴蝶蘭為具有經濟價值的園藝觀賞作物，在花卉市場頗受消費者喜愛。為了瞭解其影響花色之關鍵基因——苯基苯乙烯酮合成酶（chalcone synthase; CHS）基因，本研究利用白花紅唇蝴蝶蘭Phal. True Lady與紅花朵麗蝶蘭Dtps. Queen Scarlet為材料，進行CHS cDNA之選殖及分析。首先利用紅花朵麗蝶蘭的cDNA為模板，以蛋白質保留區設計寡核苷酸為引子，進行聚合酶連鎖反應，得到600bp、400bp、100bp三段產物，其中400bp的DNA片段經轉殖入pUC19載體內，得到5個選殖系，經核酸定序分析及轉譯成胺機酸後，與洋芜荽及矮牽牛的CHS胺機酸序列比較，顯示此五個基因片段，係源自於CHS基因。利用紅花朵麗蝶蘭所得到的cDNA片段為探針，篩選白花紅唇蝴蝶蘭之λZAPⅡcDNA庫約260,000個溶菌斑，得到11個選殖系，選取pOCH01選殖系進行全長核酸定序分析。序列長度為1.498bp，可解碼390個胺機酸、其蛋白質的分子量為42,586 Da, pI值為6.0。本研究再利用胺機酸序列比對，與其他作物比較，顯示具59.5%～64.9%的相似性。經由南方氏雜交分析，分析其基因組DNA，發現CHS基因屬於多基因族（multigene family）。

The first and key enzyme in the branch pathway of phenylpropanoid biosynthesis is chalcone synthase (CHS). It catalyzes the condensation of the acyl residues from one molecale of 4-coumaroyl-CoA and three molecules of malonyl-CoA. In order to isolate the complementary DNA sequences for CHS mRNA, degenerate oligonucleotide primers corresponding to conserved regions of chalcone synthase from various plant species were synthesized. These primers were used for polymerase chain reaction to amplify DNA fragments from first strand cDNA synthesized from poly (A)(superscript +) RNA isolated from Dtps. Queen Scarlet petals. The synthesized fragments ranged between 100 and 600bp. The sequence of these DNA fragments have been determined and their deduced amino acid sequences showed that they were derived from CHS genes. Approximately 260,000 plaque-forming units were screened from the Phal. True Lady cDNA library constructed in λZAPⅡ vector by plaque hybridization with 32P-labelled CHS fragments and 11 positive putative clones were isolated. Purified individually, Phalaenopsis chalcone synthase cDNAs were digested to draw restriction maps. One of the cDNA (pOCHS01) was chosen for sequence analysis. The coding region encodes 390 amino acids and was determined to be 42,586 Da with an isoelectric paint of 6.0. The sequence identity of Phalaenopsis CHS to other CHS polypeptide from different plant species varies between 59.5 and 64.9%. According to Southern analysis of genomic DNA digested with BamH I and EcoR I, we showed that both Phalaenopsis and Doritaenopsis chalcone synthase belong to multigene family.