馬拉巴栗(Pachira macocarpa)為木棉科常綠喬木，主要作為觀賞樹種，根據預備實驗的研究結果，發現它比其它7種常見的陸生植物含有較高的葉綠素酶(chlorophyllase, chlase)活性，而chlase是植物將葉綠素(chlorophyll, chl)轉化成脫植醇葉綠素(chlorophyllide, chlide)及植醇鏈(phytol chain)的主要酵素。因此，本研究將以馬拉巴栗為材料，利用RACE(Rapid Amplification of cDNA Ends)的技術由馬拉巴栗的葉子選殖chlase基因(PmCLH)並分析其胺基酸之功能性區間(domain)。以Real-time PCR技術分析比較所選殖PmCLH在幼葉、老葉、以及莖的基因表現量。而在蛋白質的分析方面將以大腸桿菌表現的PmCLH的重組蛋白進行兔子多株抗體的製造並以西方墨點法進行分析，並推測PmCLH在蛋白質的結構、抗原決定功能基、抗體免疫專一性、及transit peptide切除的後修飾作用。另外，也將以黃金榕葉片的LHCⅡb抗體為對照組，分析PmCLH可能存在於葉綠體的位置。之後，分別以chlase基因的全長的構築和蛋白質氨基端(N-terminal domain)部分截斷(trancate)的重組蛋白進行試管下酵素活性分析，比較將葉綠素催化成chlides的能力，以及比較chlase對chl a或chlb的酵素催化能力與受質專一性。期望能藉由chlase基因的選殖來探討chlase之結構與物化特徵，進而了解其生理功能及推測在植物葉片黃化與葉綠素分解所扮演的角色。
Pachira macocarpa, a member of the Bombaccaceae family, has been popular as ornamental plant species due to its ever-green leaves. The preliminary results show that the chchlorophyllase (chlase) activity of Pachira macocarpa is higher than the other seven plant species. Chlorophyllase, catalyzing the hydrolysis of chlorophyll (chl) to yield chlorophyllide (chlide) and phytol, is thought to be the first enzyme in the chl-degradation pathway. Therefore, we will use Pachira macocarpa as material in this study. A full-length target cDNA of chlase gene will be amplified using RACE technique followed by cloning it into the vector for sequencing, and comparing it with any known sequences in the NCBI-blastn. The response of chlase gene expressions in young and old leave as well as stem will be determined using real-time PCR. Manufactured using a rabbit polyclonal antibody and chlase recombinant proteins, pET-28a and pET-24a, will be recognized in leaves by Western blot analysis for the structure, specificity and modification of chlase. The location of chlase will be determined using LHCⅡb antibody as a control group. Both full length and trancate (N-terminal domain) of the chlase activity in recombinant proteins will be constructed, followed by catalyzing chl into chlides. In addition, the ability of catalytic activity and substrate specificity in chlase will be measured. Exploring structure and physico-chemical characteristics of chlase in Pachira macocarpa will allow us to understand physiological function in leaf and fruit in more depth.
