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標題: | 建立水稻Class I 16.9 kDa低分子量熱休克蛋自質pTS1 cDNA轉殖菸草 Establishment of a transgenic tobacco with rice class I 16.9 kDa low-molecular-mass heat-shock protein cDNA, pTS1 |
作者: | 丘鶴聲 |
出版年 : | 1995 |
學位: | 碩士 |
摘要: | 當生物體曝露在高溫時,其大部分基因的轉錄(transcription)與蛋白質轉譯(translation)皆被抑制,另有一小部分蛋白質經轉錄轉譯大量合成,即為熱休克蛋白質(heat shock proteins, HSPs)。對植物而言,15?18kDa低分子量群是熱休克蛋白質中最豐富的。 為了研究低分子量熱休克蛋白質的功能與生理角色,吾人嘗試將本實驗室先前所選殖到的水稻class I 16.9kDa低分子量熱休克蛋白質pTS1恆定表現(constitutive expression)於栽培菸草(Nicotiana tabacum cv. W38)中。pTS1全長cDNA首先以EcoRI自載體pGEM-3Z中切下,再用Klenow fragment補齊(fillin)以獲得平頭(blunt-end)insert。其次,平頭insert被接合到雙向載體(binary vector)pBI121的SmaI選殖位中,與花椰菜鑲嵌病毒35S(CaMV35S)起動子接合成融合基因。此時,吾人利用不同的引子進行聚合酵素鏈合反應(polymerase chain reaction,PCR)以放大插入DNA的片段,藉以判斷pTS1的接合方向。結果得到了編號I12為正接(sense)株,編號F5為反接(anti-sense)株。最後,將I12與F5質體分別用直接轉型法(direct transformation)轉殖入農桿菌(Agrobacterium tumefaciens)LBA4404/pAL4404,得到LI12株及LF5株。這些菌株以pTS1 cDNA為探針(probe)進行南方氏雜合(Southern hybridization)確認無誤後,準備用來感染菸草。 另一方面,吾人並進行菸草無菌培養之操作以利用葉圓片轉型技術(leaf disc transformation)轉殖菸草。經過200ug/ml的康黴素(kanamycin)篩選後,正接與反接的植株各得到七株,分別給予編號TI12-1到TI12-7與TF5-1到TF5-7,各轉殖株首先用聚合酵素鏈合反應放大篩選標(selection marker)NPT II片段以鑑定無誤後,所有植株皆進行誘導成懸浮培養系統。在誘導懸浮細胞的同時,進行了總DNA的南方氏雜合分析。吾人得到了TI12系列pTS1插入的直接證據,但TF5系列則未發現。 當懸浮細胞的生長穩定後,吾人測定其生長曲線(growth curve),選擇屬於對數生長期(log phase)的第8天大的細胞進行RNA與蛋白質免疫分析。首先以pTS1 3'端不轉譯區(3'UTR)為探針進行總RNA北方氏雜合(Northern hybridization)分析,正接細胞株被發現有分子量800nt的雜合訊號,推測其即為pTS1之轉錄產物;反接者則有1300,400,300nt大小的訊號被觀察到,這現象和反義RNA沒有明顯轉錄終點有關。吾人並使用對抗pTS1蛋白質的多株(polyclonal)抗體進行免疫轉印反應(Western hybridization),結果發現除了被施以熱休克處理的細胞外,沒有任何16.9kDa大的免疫反應點被偵測到。似乎,pTS1蛋白質的表現(expression)或累積(accumulation)量是偵測不到的。此外,無論是轉殖株或野生型的懸浮細胞,都有一個26-28kDa的免疫訊號出現,相對的葉片卻沒有見到,這可能是懸浮細胞特有的現象。 In response to the exposure of high temperature, most transcription and translation of normal proteins is repressed and a small set of new proteins (heat shock proteins, HSP) is produced in all organisms. Among them, the 15-30kDa low molecular mass (LMM) HSPs are the most predominant in plants. In order to investigate the function and physiological roles of LMM HSPs, I tried to establish a transgenic tobacco (Nicotiana tabacum cv W38) cell line with constitutive expression of pTS1, the 16.9 kDa Class I LMM HSP cloned from rice in our lab. The pTS1 full-length was first cut from pGEM-3Z with Eco RI and then filled in by Klenow fragment to produce a blunt-end insert. The blunt-end insert was ligated into the SmaI site of the binary vector pBI121 to fused with CaMV35S promoter. Then I used the polymerase chain reaction to amplify the insert region with defferent primers to get the insertion orientations. The I12 clone with a sense orientation and F5 clone with an antisense one were then obtained. I used these two clone to transform Agrobacterium LBA4404/pAL4404, and I got LI12 clone as the I12 transforming Agrobacteria clone and LF5 clone as the F5 transforming clone. These clones were identified by Southern hybridization with pTS1 cDNA as a probe. These two clones were preparing for infecting tobacco. On the other hand, I performed a procedure with tobacco plant manipulation. Then these experiments were taken together by the technique of leaf disc transformation. After 200 ug/ml kanamycin selection, there were both 7 plants obtained from these two bacteria strains, named TI12-1 to TI12-7 and TF5-1 to TF5-7 individually. Transgenic plants were identified by PCR with amplification of NPT II fragment from plant genomic DNA. Fortunately, all these named plants have passed the identification. Then all transgenic plants were induced to establish the suspension culture system. Along with the induction, the genomic DNA blotting is performed. There were direct evidences to the TI12 series, but to TF5 series were still lacking. After the stable growth of these suspension cell lines, the growth curve is to be determined. The log phase (about eight days old) cells are chosed to perform RNA and immunoblotting analysis. Northern hybridization with pTS1 3' unstranslated region (3'UTR) as the probe, there were reactive bands at 800nt, thought to be the transcript of pTS1, and 1300, 400, 300nt for the antisense lines because of the lack of suitable transcription stop sites. Using the polyclonal antibody against pTS1 protein, I was unable to obtain any reactive spots with the size of 16.9kDa from the normal temperature incubating cells of TI12 series. Therefore, it seemed that the expression or accumulation of pTS1 protein was not detectable. Besides, a 26-28 kDa immuno-responsive spot was found in every suspension cell line that does not depend on heat treatment. But it did not show up in leaves. It seemed that the 26-28 kDa spot was specific to suspension cells. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76131 |
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顯示於系所單位: | 植物科學研究所 |
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