臺灣博碩士論文加值系統

瞬間提高生長環境的溫度時，會產生熱休克反應，誘導出一系列熱休
克蛋白以分解不正常蛋白與保護細胞。 DnaK 是其中一種很重要的熱休克
蛋白，可以協助保護新合成胜的蛋白折疊或胜的移位，在生物生理活
性上扮演重要的角色。本實驗中分析 Xanthomonas campestris pv.
campestris 17 的熱休克反應時，發現 37℃ 是 Xc17 仍可穩定存活的最
高溫度。 將 Xc17 從培養溫度 28℃ 移至 33℃ 或 37℃ 時，可觀察到
至少 12 種蛋白新合成或量增加，其分子量分別約為 120，97，87.7
，80.5，78，68.4，53.1，42.3，29.1，23.4，20.2 及 18.8 kDa。在實
驗中亦發現，當 Xc17 從 28℃ 移至 33℃ 或 37℃ 時，熱休克後的最初
5 分鐘內，蛋白的合成作用減少，隨後才逐漸恢復，並有較多量的熱休克
蛋白表現。比較培養於不同培養液中的 Xc17 之熱休克反應時，發現其熱
休克蛋白的表現不盡相同。 我們從已發表的 DnaK 胺基酸序列中選取保
存性較高的兩個區域，設計兩條退化性引子，以 Xc17 染色體 DNA 為模
板，進行聚合鏈鎖反應 ( PCR )。再利用 PCR 合成的產物作為探針，
與以 ClaI 及 EcoRI 切割的染色體 DNA 所構築的基因庫進行菌落雜配。
共選殖到 5 個帶有 dnaK 基因片段的重組殖株。經過限制圖譜分析與
初步的定序，挑選其中兩個分別帶有含 dnaK 及其上游區域，以及含
dnaK 部分片段及其下游區域的殖株，進行核酸定序。利用刪減反應，
選殖適當之刪減株進行 DNA 定序。共完成 5,798 個核酸之定序，其中
包含了 5 個 ORFs。經過比對分析發現與 5 個已知的基因相似，依序為
hrcA-grpE-dnaK-dnaJ-pdxK。序列分析結果顯示，在 grpE 與 dnaK 的上
游有類似 σ32 辨認之啟動子區域 ( 在 -35 區為 CTTGAA, -10 區為
CCCA-AT )。 在 grpE 與 dnaK 之間 (nt 1254-1272)，dnaK 與 dnaJ 間
(nt 3339-3370) 及 dnaJ 的下游 (nt 4622-4648) 則各有可形成二級結
構的 inverted repeat (IR)。以 dnaK 部份片段做探針，進行北方轉漬
分析後，得到二個大小約 2.6 kb 與 1.9 kb 的轉錄產物。其中 1.9 kb
大小的訊號可能來自 dnaK 轉錄產物， 顯示，dnaK 與 dnaJ 間的 IR 可
能具有終止轉錄的功能。 而 2.6 kb 大小的訊號可能是 grpE-dnaK 轉錄
產物或 degraded dnaKJ 產物。

The heat shock response in a plant pathogenic bacterium
Xanthomonas campestris pv. campestris 17 (Xc17) was
investigated. It was found that 37℃ was the highest
temperature at which Xc17 was able to grow steadily. Cells
shifted to 40℃ showed inhibition of growth and, after a
prolonged exposure, underwent lysis. The synthesis of protein
decreased transiently following the heat shock response, and
recovered after about 10 minutes later. A shift from 28℃ to
37℃ caused increased synthesis of at least twelve proteins as
judged by SDS-PAGE. Using another culture medium XOL to grow
Xc17 for heat shock response, a protein of ~ 66 kDa was heavily
labeled. According to the molecular mass of the heat shock
protein, it is speculated that the 66 kDa protein could be a
functional homologue of DnaK. As the concentration of the
nitrogen in medium was reduced, the heat shock protein behaved
differently. It is believed that the medium used for
cultivation of bacteria in heat shock response would affect the
species and quantities of heat shock proteins induced. In this
study, the dnaK and its flanking sequences were cloned and
sequenced. Analysis of the sequence revealed three complete
ORFs and two non-complete ORFs. The deduced amino acid
sequences of the three ORFs have strong homologies to the heat-
shock proteins, GrpE, DnaK, and DnaJ. The deduced amino acid
sequence of two non-complete ORFs are homologous to the C-
terminal region of HrcA, a negative regulator of heat shock gene
expression, and the N-terminal region of pyridoxine (PN) kinase
in E. coli K12. Therefore, the gene order of these five
predicted ORFs is hrcA-grpE-dnaK-dnaJ-pdxK. The putative grpE
and dnaK promoters posses a good match to the E. coli heat-shock
consensus -35 and -10 sites for s32 binding. A potential Rho-
independent terminator was found downstream of the dnaJ. In
addition, two inverted repeat sequences located between grpE and
dnaK, as well as dnaK and dnaJ are also identified. These IR,
may be related to the transcriptional regulation, are to be
analyzed.