本研究主要是從表現序列標誌資料庫(expressed sequence tags, EST database)篩選出姬蝴蝶蘭(Phalaenopsis equestris)之花青素合成的相關基因。目前已建立之EST database乃為紅花橘心姬蝴蝶蘭花苞之cDNA library，並逢機定序後所建立之EST database。自此EST database中，我們找到五個與花色合成的相關基因，包括chalcone synthase (CHS)、chalcone-flavanone isomerase (CHI)、anthocyanidin synthase (ANS) 、flavonoid 3’-hydroxylase (F3’H)、及UDP-glucose: flavonoid 3-O- glucosyltransferase (UFGT)，其中除了CHS已得到全長外，其餘的基因則以RACE技術來得到基因序列全長，並進行多序列分析及親緣演化分析。我們利用北方雜合分析確認五個基因在四種不同花色的花苞中表現的差異性，包括紅花紅心、紅花橘心、白花黃心、以及白花白心的姬蝴蝶蘭，均為以紅花橘心姬蝴蝶蘭(P. equestris) ’W9-52’ 為母本，白花白心姬蝴蝶蘭(P. equestris) ‘W9-17’ 為父本所進行雜交後之F2子代。結果顯示這五個基因在四種不同的花色之花苞中的基因表現並無明顯的差異。為確認是否有點突變發生在CHS、CHI、ANS基因上，進而導致花色之差異，我們亦從四種不同花苞中，以RT-PCR方法增殖出CHS、CHI、ANS等基因之cDNA，經定序後分別進行核苷酸與胺基酸的多序列比對，結果發現這四種花苞中的CHS、CHI、ANS等基因，在核苷酸與胺基酸的不同位置皆有差異。進一步進行這些酵素蛋白後修飾區域的預測，發現在紅花橘心、白花黃心、白花白心之CHS胺基酸的第134-137位置有磷酸化現象，而在紅花紅心則沒有；在紅花紅心、白花黃心、白花白心之CHS胺基酸的第227-230位置有磷酸化現象，而在紅花橘心則沒有。另外，我們發現在四種不同花色之CHI與ANS基因蛋白後轉譯修飾作用，並未有差異。不過，這樣的結果仍需日後的實驗來加以確認.

This research was to study the flower color related genes from expressed sequence tags (ESTs) database involved in anthocyanin synthesis pathway in Phalaenopsis equestris. The EST database of P. equestris was established from 3,549 randomly sequenced clones of cDNA library. Five enzymes involved in anthocyanin pathway, including chalcone synthase (CHS), chlacone-flavanone isomerase (CHI), anthocyanidin synthase (ANS), flavonoid 3’-hydroxylase (F3’H), and UDP-glucose: flavonoid 3-O- glucosyltransferase (UFGT) genes were identified. The expression patterns of these five genes in four different color sets of the flower buds of P. equestris were analyzed with northern blot hybridization. Four F2 progenies of red flower-red lip, red flower-orange lip, white flower-yellow lip, and white flower-white lip derived from the F1 selfcross of the ‘W9-52’ x ‘W9-17’ were used as materials. Results showed that there was no differential expression pattern among the flower buds of the four types of P. equestris. To determine whether there is any point mutation occurred in CHS, CHI, and ANS genes among the four different flower buds, RT-PCR was carried out. Results showed that single nucleotide polymorphisms (SNPs) were observed in CHS, CHI, and ANS genes among the four different flower buds. The prediction of posttranslation modification site of each CHS, CHI, and ANS genes among the four color flower buds were also analyzed. Results showed that no differential posttranslational modification was observed in both CHI and ANS cDNA sequences. Whereas, differential phosphorylation patterns may have occurred in CHS genes of the four flower buds. However, this needs to be confirmed by further experiments.

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