臺灣博碩士論文加值系統

以UX8和LX3兩個引子(primer)，利用PCR方式合成含柑橘潰瘍病菌Xanthomonas campestris pv. citri之recX基因，長度為500 bp，經限制作用後，插入表現載體pTYB4上，得到重組質體稱為pTX5。將此質體送入大腸桿菌BLR(DE3)後，以IPTG誘導其大量表現，可純化一分子量為18 kDa之RecX蛋白。以純化之RecX蛋白注射小白鼠，得到抗RecX蛋白之抗體，提供西方墨點法分析野生株(XW47)，lexA突變株(XLE22)與recA突變株(XCK75)中RecX蛋白的產量，發現recX基因的表現受到DNA突變劑所誘導，也與lexA及recA的表現有關。為探討RecX蛋白的詳細功能，另將recX基因移至含lac啟動子之載體pFJ1上，得到之重組質體稱為pFX1。含此質體之轉形株，利用IPTG可誘導RecX蛋白的大量表現，經mitomycin C誘導後，以西方墨點法分析此菌RecA、LexA與RecX三種蛋白的產量，更證明recX基因調控lexA及recA基因的表現。以gel retardation方法分析其與DNA的作用，發現不論是recA，lexA或recX的啟動子，並不具有專一性的結合，表示RecX的調控recA或lexA，並非以repressor的角色抑制其表現。Northern blot分析得知RecX會使recA基因之mRNA之產量增加。但分析含質體pFX1之XLE22及XCK75中，RecA、LexA與RecX三種蛋白的產量則顯示RecX並不直接影響RecA或LexA之產量。而可能藉由抑制RecA之活性或干擾LexA之裂解來調控此二基因之表現，其詳細之機制則仍待進一步的實驗證明。

The expression and regulation of recX gene in Xanthomonas campestris pv. citri were analyzed by Western blotting . The result indicated that the recX gene expression was inducible by DNA damage agent. It is also demonstrated that the lexA and recA gene regulated the expression of recX gene. A RecX protein overexprssion strain was constructed by inserting the recX gene into the vector pFJ1. The function of the recX gene was analyzed by Western blotting , gel retardation experiments and Nothern blotting . Western blot analyzes indicated that the recX gene regulate the expression of the lexA gene and recA gene . Moreover , gel retardation experiments demonstrated that the RecX was not a repressor of the recA or lexA gene . Furthermore , Northern blotting demonstrated that the RecX protein increased the mRNA synthesis of the recA gene . However , Western blotting demonstrated that the RecX protein did not affect the protein synthesis of the RecA or LexA. Overall , the results from this study suggested that the RecX protein could affect the expression of recA and lexA gene by protein-protein interaction .

目　錄 pI
圖目錄 pIV
表目錄 pIV
中文摘要 p1
英文摘要 p2
壹、緒　言 p3
貳、材料與方法 p10
參、結果 p46
肆、討論 p72
伍、參考文獻 p79

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