| Summary: | 碩士 === 國立海洋大學 === 水產生物技術研究所 === 89 === Three full length cDNAs (BoCLH1, 1140 bp; BoCLH2, 1104 bp; BoCLH3, 884 bp) encoding putative chlorophyllases were cloned from the cDNA pools of broccoli floret and characterized. The deduced amino acid sequence analysis indicated that these three BoCLHs contained a highly conserved lipase motif (GHSRG), suggesting they were serine deacylase. N-terminal sequences among the three BoCLHs were more conserved than the C-terminal sequences. BoCLH2 and BoCLH3 had N-terminal sequences (Met1aVal50 and Met1aVal62) typical of signal sequence for chloroplast, whereas no typical signal sequence was identified in BoCLH1. The predicted molecular mass of BoCLH1, 2, and 3 are 34.7 kDa, 35.3 kDa, and 23.5 kDa, respectively. Genomic DNA gel blot analysis suggested that each of the chlorophyllase genes was a single copy gene and existed as a multi-gene family in broccoli genome. The RNA gel blot results showed that the mRNA transcripts of BoCLH2 and BoCLH3 were undetected in neither the floret nor the leaf during the broccoli postharvest senescence, while the mRNA transcript of BoCLH1 accumulated obviously in fresh harvested floret, 4-day-postharvested floret, and 6-days-postharvested leaf. We also used RT-PCR technique to unveil the transcription patterns of three chlorophyllase genes after ethylene treatment. In the presence of ethylene, the transcripts of BoCLH1 and BoCLH2 were induced, but the BoCLH3 mRNA was only expressed in basal level. The divers expression level and patterns of the mRNA transcripts suggested that the three BoCLHs might play different biological roles in responding to various physiological aspects in broccoli. Two alternative spliced mRNA transcripts derived from the mature mRNA of BoCLH2 were identified and they encoded two polypeptides with calculated molecular mass of 32.6 kDa and 33.3 kDa, respectively, suggesting BoCLH2 was under delicate posttranscriptional regulation.
The broccoli chlorophyllase protein was expressed in E. coli and characterized. The optimum pH of BoCLH1 and BoCLH2 was pH 8 and pH 11, respectively. The optimum temperature was 35℃ and 50℃ for BoCLH1 and BoCLH2, respectively. Both the BoCLH1 and BoCLH2 were unstable by heating to 75℃. According to amino acid sequence alignment, two conserved residues, His66 and Asp170 , were proposed to be active sites of chlorophyllase. The enzyme activities of wild type BoCLH2 and the site specific mutants of His66 and Asp170 were analyzed. The results suggested that Asp170 might play a role in the catalytic triad of chlorophyllase but not His66.
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