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|標題:||大豆種皮Flavonoid 3', 5'-Hydroxylase (F3', 5'H)基因之選殖與基因表現分析
Molecular Cloning and Expression of Flavonoid 3', 5'-Hydroxylase in Seed Coats of Soybean (Glycine max)
本論文選殖黑色種皮大豆青仁烏豆(CRWD)之色素基因flavonoid 3', 5'-hydroxylase (F3', 5'H)，供花色改造及近同源品系色素合成之分子層次研究。依據已發表物種的F3', 5'H序列設計退化引子，利用RT-PCR策略選殖含預期片段得到BG39-2殖系，再用以篩選cDNA基因庫，得到一含1,853 bp之cDNA殖系 72-1。此殖系包含1,542 bp的ORF (open reading frame)轉譯一513個胺基酸序列，並具有 59 bp的5'端非轉譯區 (UTR)及252 bp的3'端非轉譯區。經NCBI網站Blast分析結果，其胺基酸序列與矮牽牛、洋桔梗等物種的F3', 5'H序列約有46-51 %的一致性(identity)。蛋白質結構分析發現，72-1殖系分子量為57 kDa，等電點 (pI)為7.32，並具有Cyt P450等5個典型的F3', 5'H蛋白所具備之motifs，因此應為解碼大豆F3', 5'H之全長cDNA。此外，72-1殖系經上網比對時，亦與第一個發表的植物F3'H基因(Ht1)有69﹪的一致性，將72-1殖系與Ht1基因之胺基酸序列進行排比，除了在N端、C端及序列中間區域有較大變異外，其餘部分相似性頗高，且Ht1基因之分子量(57 kDa)及等電點(7.71)皆與72-1殖系相似，也包含 5個典型的F3', 5'H蛋白所具備之motifs。雖然，72-1殖系亦具有F3'H可能特有的GGEK序列，但因此胺基酸之功能與特性尚不清楚，故無法証明72-1殖系為F3'H。北方雜交分析顯示F3', 5'H在幼嫩種皮表現量最高，成熟種皮表現最低。F3', 5'H在大豆植株各組織亦以種皮的表現最為明顯，在其他組織中之表現極弱。在所有Clark近同源品系中F3', 5'H均有一 2.1 kb的轉錄信息，且以具顯性T等位基因之表現較強。南方雜交分析顯示，F3', 5'H基因在所有Clark近同源品系均為單套，且在所有隱性t基因型品系皆較顯性T基因型品系約大300 bp，經選殖顯性T基因型品系(iRT)之基因組殖系(gT殖系)與隱性t基因型品系(iRt)之基因組殖系(gt殖系)，進行序列分析、比較發現，gt殖系的intron內多出一262 bp的片段，推測t基因型應是T基因組發生插入突變 (insertional mutation)所致，至於是否因此導致二基因型間F3', 5'H基因表現的差異，則有待更深入的探討。
The blue gene, flavonoid 3', 5'-hydroxylase (F3', 5'H), was cloned from seed coats of black soybean (Chin-Ren-Woo-Dow, CRWD) for flower color engineering and molecular study on soybean isogenic lines with various pigment genes. Degenerate primers were designed according to the published sequences of F3', 5'H. An expected fragment was amplified by RT-PCR strategy and cloned as pBG39-2, a 1.8 kb full length 72-1 clone was obtained by screening a seed coat cDNA library. The 72-1 clone contains a 1,542 bp ORF which encodes a polypeptide containing 513 amino acids, a 59 bp 5' untranslated region (UTR), and a 252 bp 3' UTR region. However, the deduced amino acid sequence of the 72-1 clone showed 46 to 51 % identity to eight registered F3', 5'H proteins by Blast analysis of NCBI. The calculated molecular mass of protein encoded by 72-1 clone is 57 kDa with the isoelectric point (pI) 7.32. The predicted protein contains five typical common motifs of F3', 5'H that belongs to the Cyt P450 super family, indicating that the 72-1 cDNA clone encodes a full length soybean F3', 5'H. The deduced amino acids of the 72-1 clone shows similar molecular mass, pI and the five typical motifs of Cyt P450 of F3', 5'H as well as sequence identity (69﹪) to a recently published F3'H of petunia hybrida Ht1 gene. Furthermore, the 72-1 clone contains a putative GGEK sequence of Ht1 that was specific to F3'H. However, its function is still unknow. Functional analysis will be conducted to determine the real function of the 72-1 clone. Northern hybridization showed that F3', 5'H transcript is present mainly in seed coats at the early stage of seed development and declined as seed mature. The F3', 5'H enzymes in all the dominant T isolines are expressed stronger than those of the t genotypes. Genomic southern analysis showed that the F3', 5'H exits as a single copy gene in all the tested Clark isolines. Furthermore, the restriction fragments corresponding to F3', 5'H in all the dominant T genotypes analyzed are about 300 bp smaller than those in the recessive t genotypes. T genomic clones, gT and gt clones respectively for the dominant T genotype (iRT) and the recessive t genotype (iRt) were cloned by using PCR. It is found that an 262 bp fragment inserted at the intron of the all t genotype, suggesting that the recessive t allele may be resulted from an insertional mutation at the T allele. If the insertion caused the difference in F3', 5'H gene expression between T and t genotypes will be studied in the future.
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