Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21657
標題: Molecular Cloning and Characterization of the secE and secY genes from Xanthomonas campestris pv. campestris
十字花科黑腐病菌secE及secY基因選殖及特性的研究
作者: 江玉玲
Chiang, Yu-Ling
關鍵字: secE;轉膜蛋白;secY;pET;pQE;PCR Mutagensis;translocase;高表現載體;突變;胞外黏多醣;核心
出版社: 分子生物學研究所
摘要: 
SecY、SecE 及 SecG 所構成的核心,在菌體內負責運送蛋白之 Sec 系統中,扮演著重要的角色。 本研究主要針對其中 SecE 及 SecY 兩蛋白,從 X. campestris 菌株中進行基因選殖及 DNA 定序分析工作。 首先自本實驗室現有包含 rpoB 基因之 pBKBet-1 選殖株上游進行核酸定序工作。 定序結果發現 rpoB 上游含有一小部份 secE 基因之 3'' 端 DNA 片段及 nusG 基因片段。 為了得到完整的 secE 基因,取 Xc11 染色體 DNA 經 Sau3AI 部份切割後,回收 4-10 kb 左右的 DNA 片段構築出 Xc11 基因庫。再利用 pBKBet-1 質體以 EcoRI-XhoI 之 0.7 kb DNA 片段製成探針,經過菌斑雜交法及菌體內質體切割法,篩選到 2 個帶有雜交訊息的質體,pBKSecE12 及 pBKSecE14。 經由限制圖譜及膠體內雜交法分析,確定質體 pBKSecE14 上帶有完整的 secE 基因。接著利用次選殖株的方式進行 DNA 定序工作。 經分析後發現其中包含二個 open reading frame (ORF),若以第 296 個鹼基對的 ATG 為起始密碼,可轉譯出 136 個胺基酸 (ORF136)分子量為 15.2 kDa 的蛋白質,經電腦分析比對與 E. coli 的 secE 有 48.7% 的同質性,但若以第 330 個鹼基對的 GTG 為起始密碼,則可轉譯出 118 個胺基酸 (ORF118) 分子量為 13.4 kDa 的蛋白質,其中 ORF118 包含在 ORF136 之內,同樣與 E. coli 的 secE 有 48.7% 的同質性。 第二個 ORF 則起始於第 700 個鹼基對,可轉譯出 186 個胺基酸,類似 E. coli 的 NusG 蛋白。 此外,經電腦分析結果顯示 ,ORF136 所轉譯出的蛋白與 E. coli 之 secE 相似,可產生三個轉膜區域但沒有可被胜辨識切割的位置; 而 ORF118 所轉譯出的蛋白則只能產生 2 轉膜區域,且有被胜辨識及切割的切位。 利用引子延伸法分析,可以在 ORF118 之 GTG 起始密碼上游 29 個鹼基對找到轉錄起始點,並可找到類似 E. coli 之 -10 及 -35 啟動子辨識區域。 因此,初步認定 Xc11 之 secE 可能以 GTG 為起始密碼,並有 2 個轉膜區域。為得到大量的 SecE 蛋白,將 secE 基因黏接到高表現載體 pET21bT 中,利用 IPTG 誘導表現後回收蛋白,將進一步用以製作抗體,以利於進行西方墨點法偵測。此外,為了解 secE 在 Xc11 中所扮演的角色,利用 PCR mutagensis 的方式產生含 secE基因 3'' 端部份的 DNA 片段,並黏接在質體 pBK-CMV 上,而構築出 Xc11 secE 基因突變基因庫,並利用單一交換將 Xc11 染色體上之 secE 基因互換成突變株。 我們並對 rpoA 基因上游區域,利用部份刪除法進行核酸序列分析中,在所完成定序的 3158 個鹼基對中,共找到 6 個 ORFs,經由與 E. coli 比對,得到轉譯出蛋白順序為 L30-L15-SecY-S13-S11-S4,其中 secY 起始於第 578 個鹼基對終止於第 1945 個鹼基對,可產生 455 個胺基酸,分子量為 48.4 kDa與 E. coli 的 SecY 有 56.3% 的同質性,並預測其可能含有與 E. coli 相類似的 10 個轉膜區域。 由於完整的 SecY 蛋白難以大量表現,故利用部份片段的 secY 基因,黏接到高表現載體 pET32a 中,希望能藉由此方式而達到蛋白的大量表現。

The Sec system is the major pathway required for the secretion of proteins in many bacteria. It has been showed that the core enzyme of Sec system is composed of SecY, SecE and SecG. The goal of this study is to clone and characterize the secE and secY from Xanthomonas campestris pv. campestris (Xc11). The nucleotide sequence of the previously isolated pBKBet-1 plasmid that containing the upstream region of the Xc11 rpoB was determined. Results of the DNA sequence analysis revealed that the genomic location of the secE may be immediately upstream of the rpoB operon and have the same gene order as that in E. coli. By using the EcoRI-XhoI 0.7 kb fragment of pBKBet-1 that containing nusG and part of the secE as probe, a recombinant l phage that bearing the intact secE gene was isolated from the constructed Xc11 genomic library. In vivo excision was performed to generate pBK-CMV derivated phage from the recombinant l phage and designated as pBKSec14. The nucleotide sequences on both strands of the 1.2-kb insert were determined. Results of the DNA sequence analysis revealed two open reading frames (ORF). The first ORF (ORF136) begins at nt 296 and ends at nt 686, from which a 136 amino acids with molecular weight of 15.2 kDa could be translated. A GTG codon at nt 329 was suggested as the start codon of the same ORF by primer extension analysis. This ORF (ORF118) encodes a 118 amino acids protein with a molecular weight of 13.4 kDa. Both of the ORF 136 and ORF118 showed 48.7% amino acid sequence identities with that of the E. coli. The protein encoded by ORF118 has been predicated to have two transmembrane domains (TMs) and has a signal peptidase cleavage site at the 21st amino acid. The second ORF (ORF186) begains at nt 700 and ends at nt 1257 from which a 186 amino acids could be encoded. To overexpress SecE protein, the secE gene was PCR amplified and subsequently cloned into pET21bT vector. The SecE with six histidine amino acids at its C-terminus was purified through His-bind resin. The purified SecE would be used as an antigen to produce antiserum for Western blot analysis. Moreover, the DNA sequence immediatedly upstream of the S13 gene of the Xc11 rpoA operon was also determined. Results of the 3158-bp DNA sequence analysis revealed that there are six ORFs (L30-L15-SecY-S13-S11-S4) and have the same gene order as that of the E. coli. The ORF455 begins at nt 578 and ends at nt 1945, from which a 455 amino acid with molecular weight of 48.4 kDa could be translated. The Xc11 SecY showed 56.3% identity with the E. coli SecY protein. Unfortunately, the protein encoded by the full length DNA fragment of secY gene can not be overexpressed in E. coli.
URI: http://hdl.handle.net/11455/21657
Appears in Collections:分子生物學研究所

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