| Summary: | 碩士 === 中國文化大學 === 生物科技研究所 === 97 === Chlorophyllase (chlorophyll-chlorophyllido hydrolase, Chlase; EC 3.1.1.14), which catalyzes the hydrolysis of Chl to yield chlorophyllide and phytol, is thought to be the first enzyme in the Chl-degradation pathway. The objectives of this study were to clone the Chlase gene from Pachira macrocarpa, explore its molecular structure and physico-chemical characteristics, and understand its physiological function in leaf and fruit.
Two Chlase Pachira macrocarpa genes PmCLH1, PmCLH2 were cloned using RACE(Rapid Amplification of cDNA Ends). The full-length cDNA of these two clones are 1189 and 1309 bps, respectively, and they can be translated into 339 and 314 amino acids, separately. PmCLHs RNA was not transcriptionally induced in all tissues. The expression level of PmCLH1 RNA in young leaf was significantly higher than that of old leaf and stem . However, PmCLH2 gene transcripts in old leaf were significantly higher than young leaf and stem. Expression of the PmCLH1 gene was significantly induced in young leaf compared to PmCLH2 gene.
Manufactured using a rabbit polyclonal antibody and PmCLH1 recombinant protein were clearly recognized in leaves by Western blot analysis, compared to PmCLH2. PmCLH 1 antibodies can be recognized by the three major signals in the young leaves and one major signal in the old leaves at molecular weight 31 kDa. PmCLH1 may exist in the chloroplast of the inner membrane using LHCⅡb antibody as a control group. Four kinds of the PmCLHs enzyme activity in recombinant protein were constructed, and constructs could catalyze Chls into the Chlides. In addition, the ability of catalytic Chl b activity and substrate specificity in PmCLH1was significantly higher than that of in PmCLH2. Although the amino acid sequence in PmCLHs is similar, the specificity of antibodies between PmCLHs can be distinguished. Therefore, we speculate that PmCLHs in the three-dimensional structure of proteins are different due to the various antigens in the function base and immune specificity of PmCLHs. PmCLH1 in the old leaf was removed after the transit peptide modification. However, most of PmCLH1 in the young leaf were not modified after the completion of the addition.
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