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標題: | 線狀噬菌體Cf2免疫基因之純系化 Molecular Cloning of the Immunity Gene of Filamentous Phage Cf2 |
作者: | 蔡宜芳 |
出版年 : | 1985 |
學位: | 碩士 |
摘要: | 當柑橘潰瘍病病菌(Xanthomonas compestris pv. citri)被Cf2感染後,被感染的細菌(XW47′)不再被Cf2感染。Cf2是線狀噬菌體亦具此免疫性甚?特殊,故本論文除對此免疲性加以探討外,更將免疫基因加以純系化。 線狀噬菌體Cf2感染過的寄主(XW47′)對Cf2有免疫力但Cf2仍可吸附在XW47′上,且單鏈的噬菌體DNA也可進入XW47′而形成雙鏈的複製型DNA。 ?了純系化(clone)Cf2控制免疫的基因,首先定出五個核酸限制?,EcoRI.BamHI.KpnI.pvuII.HindIII對Cf2基因體之切割位置。由於這幾個酵素對Cf2基因體只有1?2個切割位置,因此並不適用於純系化免疫基因,故進一步做限制?Sau3AI切割位置的定位。Cf2基因體被Sau3AI切割所產生的片段多於二十個,不能用一般方法來作物理拼圖,所以先以Sau3AI部份切割Cf2DNA產生的片段以pBR325當媒介體(Vector),送入大腸菌(RRI strain)加以純系化。再以轉形株內質體DNA所含的Cf2DNA片段重疊之情形定出Sau3AI切割位置。 進一步,轉形株藉由GJ23內質體的作用,將pBR325及其夾帶的cf2DNA由大腸菌送入柑橘潰瘍病菌,而在柑橘潰瘍病菌內建立起噬菌體cf2的基因庫,柑橘潰瘍病菌藉此方法接受質體的比例約在0.2×10-5?4×10-5之間。 最後著手尋找控噬菌體cf2免疫力的基因之所在,確實找到二株結合株,抑制噬菌體cf2形成溶菌斑。 After Xanthomonas campestris pv. citri was infected by Cf2, the infected cell could not be superinfected by Cf2. It is so special that Cf2 was a small filamentous phage, but still has its own immunity system. The immunity of this phage was studied and the gene involved in the system was cloned. Infected cell was immune to Cf2, but Cf2 could adsorb on these cells. Phage DNA could enter into infected cells and replicated into replicative form (RF). In order to clone the immunity gene, the physical map of Cf2 RF DNA was constructed. The first physical map was constructed with five restriction endonuclease Eco RI, Bam HI, Kpn I, Puv II, and Hind III. There are only one or two cutting sites of those restriction endonucleases on Cf2 DNA. The DNA fragments cut by these enzyms were too long to be used to clone the immunity gene. Therefore second physical map was constructed with restriction endonuclease Sau 3AI. Cf2 DNA was cut into twenty fragements when it was digested by Sau 3AI. Since the number of fragements is too many, we could not use the normal method to construct the physical map, therfore Cf2 DNA was partially digested by Sau 3AI then, using pBR325 as vector. These fragements producted by partial digestion were cloned into E. coli. The plasmid DNA from tramsfprmants was isolated and the physical map was constructed by the overlapping condition of the Cf2 fragements present in transformants. With the help of the plasmid of GJ23, the pBR325 containing partially digested Cf2 DNA was transfered from E. coli to X. campestris pv. citri. The ratio of conjugation was between 0.2 × 10-5 and 4 × 10-5. From 50 conjugants, 40 conjugants were selected for analysis of the present of immunity gene. Two conjugants were immune to Cf2 infection. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75530 |
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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