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標題: | 菸草基因組澱粉磷解?基因的選殖與轉殖 Cloning and Transformation of Tobacco Genomic Starch Phosphorylase Gene |
作者: | 徐玉清 |
出版年 : | 1992 |
學位: | 碩士 |
摘要: | 高等植物澱粉磷解?催化澱粉的分解(釋出G-1-P)與合成(釋出Pi ) 反應,但是在什麼情況下進行分解、什麼情況下進行合成,則仍不清楚,因此最根本的方沃便是直接由植物基因組澱粉磷解?基因著手,研究該基因在植物體內如何表現。本實驗先用馬鈴薯幼嫩塊莖L型澱粉磷解?cDNA的0.7kb HindⅢ片段作為探針,由菸草基因組的基因庫中,篩選出8.1kb的BamHI片段。經過選殖、次選殖及核酸定序,找到可能包含promoter與轉錄起始部位的1.4 kb BamHI • PstI片段。在該片段的後面接上GUS報導基因(在pBI101.1中),經瘤農桿菌送入菸草,已得到健康的轉殖植物。由南方氏墨點法及MUG染色,知道該片段的確含有promoter,並確實併入菸草的基因組中,在葉與根部表現。另外從8.1kb的BamHI片段中,截取3.7kb的BglⅡ• EcoRI片段及2.5kb 的KpnⅠ• SmaⅠ片段,反向接在caMV 35S pr○mter(在pMON530中)後面,經瘤農桿菌送入菸草。後者已得到健康的轉殖植物,目前移至溫室種植於盆土;前者則因小苗黃化,重新處理一次,現已移出生長室種植於根基旺培養土。因為8.1kb BamHⅠ片段與.馬鈴薯cDNA相較,下游部分還少了大約400個發基對,所以仍繼續篩選其他基因庫,以找出完整基因。 Starch phosphorylase is one of the important enzymes involved in starch metabolism. Starch phosphorylase catalizes the phospholysis of α-glucan and releases glucose-1-phosphate as a degrading product. The enzyme also catalizes the reversal reaction by that glucose-1-phosphate consumed and starch synthesized. In plants, there are two types of starch phosphorylase, typeⅠand type Ⅱ, which are located in the cytosol and in the plastid, respectively. The biochemical characteristics of phosphorylase have been studied extensively. In contrast, we do not know much about the physiological roles of phosphorylase in starch metabolism. To learn more about starch phosphorylase, we cloned and sequenced one genomic gene of tobacco starch phospho-rylase. By using a cDNA clone of potato L form starch phosphorylase as a probe, one 8.1 kb genomic clone was screened from a λ40W38 BamH Ⅰgenomic library. The genomic clone was subcloned and sequenced. To compare the sequences of tobacco genomic clone with potato L form cDNA, 11 segments of the tobacco 8.1 kb clone showed a high homology with potato cDNA. Besides, the 8.1 kb genomic clone seemed not be a complete gene and it missed about 400 bp of the 3’ region. A 1.4 kb fragment of 5’ end of the tobacco 8.1 kb genomic clone was found to contain the promoter and the initiation site of the coding sequence. The 1.4 kb promoter-containing fragment was ligated with the GUS reporter gene of pBI101.1 vector. The construct was transformed into tobacco mediated by Agrobacterium infection. Several transformants were analyzed by Southern blot and by assaying GUS activity. Based on the expression of the GUS gene in the roots and leaves of transformants, we believed that the examined 1.4 Kb fragment of starch phosphorylase gene contained the promoter activity. In order to produce antisense RNA to block the expression of the endogenous starch phosporylase gene, a 3.7 Kb BglⅡ•EcoRⅠfragment and a 2.5 Kb KpnⅠ•SmaⅠfragment were isolated from the structure sequence of the starch phosphorylase genomic clone, and these two fragments were ligated into pMON530 in antisense orientation, pMON530-sp3.7BE and pMON530-sp2.5KS. These constructs were transformed into Agrobaterium and infected wild type tobacco. The transformants of pMON530-sp3.7BE and pMON530-spKS are growing and going to be analyzed. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75871 |
全文授權: | 未授權 |
顯示於系所單位: | 植物科學研究所 |
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