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Tissue-specific characterization of a wound-induced protein WI12 in Mesembryanthemum crystallinum
Cell wall protein
本論文原擬篩選耐鹽模式植物冰花 (Mesembryanthemum crystallinum L.) 之第五群抗病相關蛋白 (PR-5) 之基因，故構築冰花癒傷組織之cDNA基因庫，並以PR-5之N端與C端保留區之序列設計引子，利用所得之RT-PCR產物進行篩選，初步獲得5個cDNA clones (編號MC1至MC5)，其中並無PR-5基因，經評估後決定對其中一個與傷害有關的基因MC1深入研究。
MC1全長993 bp的包含360 bp的轉錄區域，編碼蛋白含119個胺基酸，分子量12,683 Dalton。推衍之胺基酸序列與馬鈴薯的傷害誘導蛋白WUN1的相似度達79%，故命名為WI12。由南方墨點分析及與EST中3個似WI12片段比對結果均顯示冰花基因組中含有2個WI12。WI12基因的表現受傷害、真菌感染與methyl jasmonate誘導。WI12在4週大年輕葉受傷後3h達最大表現量，而在8週大成熟葉受傷後24h才達最大表現量，鹽處理4週大冰花以誘導CAM (景天酸代謝) 轉換，其葉部受傷後表現WI12基因的時間與8週大成熟葉相似，證實CAM轉換會影響WI12表現的時間。原位雜交的結果顯示WI12基因在韌皮部均為持續性表現，在葉肉細胞則會被傷害與MeJA所誘導，而在胎座與種子的表現則會受發育時期的調控而改變其表現量或位置。推論二個WI12基因的其中一個是持續表現型的基因，其具有韌皮部的組織專一性，不受傷害或MeJA所誘導；另一個 WI12基因則是會受傷害與發育時期的調控，主要表現於葉肉細胞、胎座與種子。
Abstract The original goal of this thesis was to clone a PR-5 gene of common ice plant (Mesem-bryanthemum crystallinum L.). A cDNA library was constructed from calli of ice plant and screened the cDNA library with a RT-PCR product that was amplified by degenerate primers designed according to the conserved N- and C-terminal sequences of PR-5. Five cDNA clones (MC1 to MC5) were obtained and sequenced, but the gene for PR-5 was not found in these clones. We decided to focus on one of the clone MC1, a wound-inducible gene, to re-veal the possible function of this wound-induced protein in this facultative halophyte. The MC1 cDNA is 993 bp in length, which contains an open reading frame encoding a protein of 119 amino acids with a molecular weight of 12,683 dalton. The deduced amino acid sequence is similar to a wound-induced protein WUN1 with 79% similarity. Therefore, it's named WI12. According to the result of southern blot and comparison with 3 WI12-like DNA fragments in EST, there are at least two WI12-like genes in the genome of ice plant. Wounding, pathogen infection and exogenous methyl jasmonate treatments induced the ex-pression of WI12 gene. In 4-week-old young leaves, the expression of WI12 reached the maximal level at 3h after wounding. In 8-week-old mature leaves, the maximum expression level of WI12 was detected at 24h after wounding. The time course of WI12 expression in salt-stressed young leaves was similar to that of the mature leaves. These results showed that the switch from C3 to Crassulacean acid metabolism in young ice plant had great influ-ence on the expression of this wound-induced gene. The results of in situ hybridization showed WI12 were constitutively expressed in phloem and induced by wounding and MeJA in mesophyll cells. Furthermore, the expression of WI12 was under developmental control in placenta and seeds. The results strongly suggested that one of two WI12 genes was con-stitutively expressed with a tissue specificity in phloem, and it was not induced by wounding and MeJA. Another WI12 was regulated by wounding and development and expressed in mesophyll, placenta and seeds. The anti-WI12 antiserum, raised by WI12 protein overexpressed in E. coli, was used for immunostaining and immunolocalization studies of WI12. The results of immunostaining showed that the locations of WI12 protein accumulation were parallel to RNA, but they were not accumulated to the same levels. The comparison between the results from in situ and immunostaining suggested that the accumulation of WI12 was regulated at translational and/or post-translational level. In wounded stems and leaves, the deposition of callose at the same tissues as WI12 accumulation. The results of immunolocalization showed WI12 is a cell wall protein, where accumulates preferentially in primary cell wall and extracellular ma-trix. Based on the accumulation sites in cell wall, we suggested the function of constitutively expressed WI12 might be related to the cell wall development in phloem. The wound-induced WI12 might be involved in the reinforcement of cell wall composition after wound-ing and, as the result, increase the ability to defense insects and pathogen attach.
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