| Summary: | 碩士 === 國立中興大學 === 農業生物科技學研究所 === 88 === 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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