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研究生:張家銘
研究生(外文):Chang, chai-ming
論文名稱:Xanthomonascampestrispv.campestris合成胞外多醣所需基因orf482之特性
論文名稱(外文):Molecular characterization of orf482 required for the synthesis of Xanthan polysaccharide in Xanthomonas campestris pv. campestris
指導教授:曾義雄, 溫福賢
指導教授(外文):Yi-Hsiung Tseng, Fu-shyan Wen
學位類別:碩士
校院名稱:國立中興大學
系所名稱:植物學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:1998
畢業學年度:86
語文別:中文
論文頁數:3
中文關鍵詞:胞外多醣跳躍子
外文關鍵詞:Xanthan polysaccharideTn5(pfm)CmKm
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中文摘要: 革蘭氏陰性菌Xanthomonas campestris pv. campestris會分
泌大量的胞外多醣,為十字花科蔬菜的黑腐病原菌。在工業上此菌卻是生
產xanthan gum的菌種。因此本實驗室對其合成xanthan gum的基因與調控
感到興趣。先前本實驗室曾利用含有跳躍子的質體pUT-Tn5(pfm)CmKm使野
生株Xc17突變,得到一些胞外多醣合成缺失的突變株。經過研究發現這些
突變株缺失的基因座落於Xc17染色體上八處位置,之後將其命名為eps1、
eps2、eps3、eps4、eps5、eps6、eps7和eps8等基因座。本實驗所探討的
是eps5基因座上的胞外多醣突變株AY61E。為了選殖AY61E上所突變的基
因,Xc17的基因庫中進行Southern hybridization篩選,得到一株 pEXO40
重組cosmid帶有eps5基因。由此pEXO40中,經次選殖得到一重組質體
pR2-2能將AY61E互補回黏濕型菌落。經由核酸定序的結果發現,pR2-2之
insert帶有一open reading frame ORF482 (主導482個amino acids,Mr
為54,013) 與X. campestris pv. campestris gene 1蛋白有99% 的相似
性。經由Northern hybridization分析發現與orf482的基因序列大小相符
的轉錄產物(1.5 kb)被偵測到。以primer extension的實驗在orf482上游
發現有轉錄啟始點。在orf482上游的一458 bp 的PstI DNA片段接入啟動
子選殖載體FY13-9中,結果能有效表現出載體上的lacZ報導基因。以E.
coli S30 in vitro transcription/translation system分析,所獲得之
蛋白產物與預測的ORF482蛋白大小相符。ORF482的C terminal amino
acids與Osbourn et al. (1990) 發表的 gene 1蛋白(regulatory
protein) 序列有99%的同質性。對於 orf482是否參與gum operon啟動子
的調節,發現在帶有gum promoter的啟動子選殖載體轉形到AY61E與Xc17
中其-galactosidase活性表現相似。AY61E對於chloramphenicol及鹽的
耐受性較高。在胞外多醣含量的測試結果顯示,AY61E其胞外多醣含量約
為Xc17的29.6%,而互補株61E(pR2-2)的胞外多醣含量能恢復到Xc17
的54.6%。在致病性的測試上,AY61E對白菜葉的致病力減弱,對於小白菜
幼苗的致病性也只有Xc17的11%。對於orf482基因的轉錄方向上經實驗證
實在Xc17染色體圖譜中為逆時鐘方向轉錄。


Abstract: The gram-negative bacterium Xanthomonas campestris pv.
campestris is an important plant pathogen, causing black rot in
crucifers worldwide resulting in tremendous loss. It is also a
producer of xanthan gum, a substance which has a variety of
applications in petroleum production, cosmetics and food
industries. We were interested in expression and regulation of
the genes involved in xanthan gum synthesis. We have
previoumutagenesis. Using pulsed-field gel electrophoresis
(PFGE) and Southern hybridization, the mutations leading to the
non-mucoid phenotypes have been located at eight loci in the
Xc17 chromosome, which were named eps1, eps2, eps3, eps4, eps5,
eps6, eps7 and eps8, respectively.
This study focused on the non-mucoid mutant AY61E. One
recombinant cosmid, pEXO40, was cloned from the genomic bank of
Xc17. After deletion mapping, a smallest clone, pR2-2, was
obtained which contained a 2.2-kb insert. Mucoid phenotype was
restored to AY61E harboring pR2-2, indicating that the pR2-2
insert contained the gene responsible for the mutation in AY61E.
Nucleotide sequence analysis of the pR2-2 insert revealed an
open reading frame (orf482) able to encode a polypeptide with a
calculated molecaa weight of 54,013 dal. The deduced amino acid
sequence of the C-terminal 153 residues of ORF482 exhibited 99%
identity to the gene 1 of X. campestris pv. campestris strain.
In Northern hybridization of the total RNA extracted from Xc17,
only one transcript with the size similar to that of the orf482
coding region (1.5 kb) was detected. Primer extension indicated
the transcription start site to be the base T at 232 nt upstream
from the orf482 codon region. A 458-bp PstI fragment before the
orf482 start cod was found to efficiently express the lacZ
reporter when cloned as a transcriptional fusion unit in the
promoter-proving vector pFY13-9. Expression of the pR2-2 insert
in vitro using E. coli S30 transcription/translation system
produced a protein of 54 kDa which is similar to the MW deduced
from nucleotide sequence. The promoter of gum operon, a cluster
of 12 genes required for xanthan synthesis, cloned as a
transcriptional fusion expressed normally in AY61E indicating
that its expression is not regulated bORF482. AY61E exhibited
higher resistance to chloramphenicol and salt. The amounts of
the xanthan gum produced by AY61E and AY61E(pR2-2) were about
29.6% and 54.6%, respectively, of that produced by Xc17. AY61E
showed attenuated virulence in causing black rot in chinese
cabbage and pakchoi. The transcription direction of orf482 was
determined to be counterclockwise on the Xc17 chromosome map.


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