Abstract
欲建立奇異球菌( Deinococcus )之基因選殖系統必須先進行選殖載體之 構建及分離可接受載體的無質體且具低同源重組能力之菌株。本實驗中 D. radiodurans IR 經由 MNNG 突變處理後所分離到一輻射敏感株 D. radiodurans PF3-3 及本實驗室先前所篩得之另一 D. radiodurans IR輻 射敏感株 D. radiodurans S101, 經證實均無原 IR 菌株所擁有之28 kb 質體 pST7。然而 S101 菌株的 DNA 轉形能力較 IR 菌株高約 1000 倍, 但 PF3-3 菌株之 DNA 轉形能力卻與 IR 相近。S101 菌株與 PF3-3 菌株 之輻射抗性自發恢復頻率為 1-5 X 10^-8, 顯示質體 pST7 在 IR 菌株高 輻射抗性之產生並非扮演必要的角色。我們將帶有可於 D. radiodurans 中表現抗藥性的 cat 基因之 Escherichia coli 質體 pBR328 與 IR 質 體 pST7 以不同限制脢處理再行接合後所得之重組質體, 經轉形發現一 33 kb的重組質體 pIR7 於 E. coli 中以不穩定狀態存在, 且其再轉形 E. coli之頻率較其他重組質體低約 500 -- 1000 倍, 而此特性與重組質 體之大小無關。上述的 pST7 與 pBR328 的重組質體中, 亦只有 pIR7 可 轉形 IR菌株並篩得 Cmr 轉形株, 而這些轉形株中原 pST7 質體已消失且 均可被偵測到一 6.6 kb 之質體存在, 此質體我們稱之為 pCMR。我們推 測 pCMR 乃為 pIR7 與被轉形 IR 之質體 pST7 作序列重組合而產生, 而 此質體可能帶有於 IR 菌株中存在所需之複製因子及作為篩選用的抗藥性 基因 cat,且由轉形實驗顯示 pCMR 能以高轉形頻率穿梭於 PF3-3 菌株、 S101 菌株及另一 D. radiodurans 菌株 R1 間而得到 Cmr 轉形株, 並且 不改變其型態及存在的穩定性。但當 pCMR 轉形 E. coli 後, 卻無法由 E. coli 中再分離到完整的 pCMR, 而所得的是各種可能被 E. coli 行刪 除作用而來的多態型刪除質體, 其中唯一能表現 cat 基因之重組質體為 5.4 kb 的 pCM5, 然而 pCM5 卻無法再轉形入各個 D. radiodurans 菌株 。由以上結果看來, pCMR 有被發展為 D. radiodurans 基因選殖系統選 殖載體之潛能且 PF3-3 菌株亦可作為質體轉形之接受株。另外我們也證 明 D. radiodurans 與 E. coli 質體轉形間存有的限制障礙。 Cloning vector construction and a plasmid-free recipient isolation must be accomplished for developing the gene cloning system in Deinococcus. Two radiation-sensitive mutants, D. radiodurasns S101 and D. radiodurans PF3-3, were isolated from MNNG-mutagenized D. radiodurans strain IR. Both also cured the 28 kb pST7 which is the cryptic plasmid of strain IR and the spontaneous reversion frequencies of them to radiation resistance were 1--5 x 10^-8. This indicated that the plasmid of strain IR was not necessary for radiation resistance. The native transform -ability of strain S101 was 1000-fold higher than IR's, and strain PF3-3 was similar to strain IR's. An Escherichia coli plasmid pBR328, that confers Cmr ( chloramphenicol ) resistance, was ligated with pST7 to yield the 33 kb hybrid plasmid, pIR7. pIR7 transformed various E. coli strains at low frequencies and existed unstably in E. coli strains. When pIR7 transformed strain IR, the pST7 disappeared and only a 6.6 kb recombinant plasmid, designated pCMR, could be detected in these Cmr transformants. We assumed that pCMR was generated by rearrangement between pIR7 and pST7. pCMR was able to transform D. radiodurans plasmid-free strains, strain S101 and strain PF3-3, to Cmr at high frequencies and remained unchanged through these transfers. Various deleted plasmids were isolated from E. coli which transformed with pCMR. Only pCM5 (sized 5.4 kb) of deleted plasmids demonstrated to be Cmr, but it could not transform D. radiodurans strains any more. Conclusions of above results, (i) pCMR should carry the replication element of pST7 besides the selection marker, cat gene, and it is potential to be a cloning vector for D. radiodurans; (ii) there were some barriers existed in plasmid transformation between D. radiodurans and E. coli; (iii) the plasmid-free strain,strain PF3-3, might be an appropriate recipient for plasmid transformation in D. radiodurans; (iv) the plasmid of D. radiodurans IR was not necessary