利用轉殖菸草研究藕蛋白基因?動子表現的調控和大豆成熟蛋白的生理角色

Abstract

本篇論文主要利用轉殖菸草研究藕蛋白（lotamin）基因?動子（promoter）的調控，以及轉殖會大量累積大豆Lea蛋白的植株，進行這些蛋白質的組織定位及其生理功能分析。在藕蛋白基因?動子方面，轉殖三段不同長度的藕蛋白基因上游DNA片段於pBI 101質體的報導基因（GUS基因，β-glucuronidase）上游，分別為pNEL 819含819bp、pNEL 498含498 bp和pNEL 300含300 bp，皆以NPT II基因表現抗kanamycin作轉殖的篩選。篩選出轉殖成功的植株，並以PCR確認含GUS基因。從種子的kanamycin抗性發芽試驗，發現抗性能力的遺傳符合孟德爾遺傳律，表示轉殖基因穩定插入核染色體。從種子的萃取液作GUS螢光活性測定，pNEL 819的轉殖株GUS活性最高達419.870 U（pmole MU/min•mg protein），pNEL 498的轉殖株GUS活性最高達34.60 U，pNEL 300的轉殖株GUS活性最高達37.95 U。GUS組織化學分析，pNEL 819的轉殖株表現在莖的髓部，和維管束交接的薄壁細胞，另外在花梗的髓部，子房和果莢的中軸，以及部份種子的胚也被偵測到。pNEL 498亦有類似的表現情形，顯示更上游區域或其他區域應有對組織特異表現的調控區域。在大豆Lea蛋白方面，轉殖三個不同lea基因，GmPM 1、GmPM 2和GmPM 8的cDNA取代質體pBI 121的GUS基因，構築於CaMV 35S?動子下游，這三個構築質體分別為pGMZ 2、pGMZ 16和pGMZ 238。在pGMZ 2的轉殖株，以Western blotting確認12棵轉殖株能表現GmPM 1蛋白，其中三棵的蛋白含量較高。分離葉片葉綠體，發現大部份蛋白質留在高速離心的上清液，僅少量蛋白留在純化的葉綠體內。在pGMZ 16的轉殖株，以Western blotting確認八棵會表現GmPM 2蛋白，其中二棵蛋白含量較高。pGMZ 238以PCR找到十棵轉殖株含有GmPM 8 cDNA，其中二棵植株矮化，可能是根系發育不良的結果，以Western blotting分析發現此二棵植株的GmPM 8蛋白含量確實較高，是否因為該蛋白質的大量累積，才造成植株矮化，則有待進一步分析。

The goal of this thesis is to study the regulation of expression of lotamin promoter and the expression of soybean Lea protein by transgenic tobacco. Three DNA fragments of lotamin promoter with 819 bp, 498 bp and 300 bp respectively, were inserted into the upstream of promoterless GUS gene of pBI 101 plasmid. These constructs were called pNEL 819, pNEL 498 and pNEL 300 respectively. Transformants of these constructs were screened by kanamycin resistance property and were further confirmed that they contain GUS gene by PCR. Results from seed germination test show that kanamycin resistant inheritance follows the Mendel's law. This pneomenon indicates that transgenic genes were stably inserted into nuclear chromosome. Seed proteins extracted from transformant with pEL 819 or pNEL 498 or pNEL 300 were used in GUS fluorometic assay, and activities up to 419.78U, 34.60U, 37.95U were detected. In GUS histochemical analysis, transformants with pNEL 819 constract express GUS activity in pith of stem, parenchyma cell near vascular bundle. Such activity also shows in other tissue, including pith of pedicle, central axis of ovary and fruit and embryo of seed. Transformants with pNEL 498 shows similar GUS expression pattern of pNEL 819. This result reveals that further upstream or another region of lotamin promoter might participate in regulating lotamin expression. In soybean Lea proteins study, three constructs, pGMZ 2, pGMZ 16, pGMZ 238, were pBI 121 with cDNA of different soybean genes - pGmPM 1, pGmPM 2, pGmPM 8. 12 transformants with pGMZ 2 construct can express GmPM 1 protein confirming by Western blotting, and three of them can experss GmPM 1 protein in higher level. Analysis of isolated chloroplast of leave shows that the majority of GmPM 1 protein retains in 10000xg supernatant. In the case of transformants with pGMZ 16, 8 transformants can express GmPM 2 protein and two of them shows in higher expression level. 10 transformants with pGMZ 238 constract were confirmed by PCR, and their GmPM 8 protein was detected on Western blotting. Two transformants express GmPM 8 protein in larger qualitity and present abnormal morphology.