迟缓爱德华氏菌基因组fosmid文库的构建、opp基因簇的克隆及aroA基因缺失突变株的构建

IOCAS-IR > 海洋环流与波动重点实验室

	迟缓爱德华氏菌基因组fosmid文库的构建、opp基因簇的克隆及aroA基因缺失突变株的构建
其他题名	Construction of Edwardsiella tarda genomic fosmid library ,cloning of opp gene cluster and construction of Edwardsiella tarda aroA in frame-deletion mutant
	杨佳银
学位类型	博士
	2008-06-15
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	迟缓爱德华氏菌 Fosmid文库 Kegg 毒力相关基因寡肽透过酶 Opp Aroa 基因缺失减毒活疫苗
摘要	本研究构建了迟缓爱德华氏菌(Edwardsiella tarda)LSE40 基因组fosmid文库，该文库共包含2 500个克隆，插入片段平均大小为33.6kb，文库总容量约84Mb，覆盖E.tarda LSE40基因组（按5Mb计算）超过16倍。随机挑取1 000个fosmid克隆进行双末端测序共得到1 741条高质量的序列，序列平均长度546 bp，全长949 997bp，约为E.tarda基因组的19％。将这些序列提交到KEGG自动注释服务器KAAS对所得序列进行代谢途径分析，得到E.tarda LSE40的KO (KEGG Orthology) 注释。分析结果表明，与代谢途径相关的基因有932条序列，与环境信息处理相关基因283条，与遗传信息处理相关基因220条，与细胞进程和人类疾病相关的基因分别为64条和16条。同时将序列进行BlastX，按照微生物致病性共同主题找到61个毒力相关基因。Fosmid文库的建立和部分基因组序列的生物信息学分析为进一步研究E.tarda LSE40的致病机制、代谢机制和生理生态机制提供了丰富的物质基础。通过比较基因组的方法，从E.tarda LSE40 fosmid文库克隆到编码寡肽透过酶的opp基因簇，该基因簇全长6 741bp，含有5个ORF，依次编码OppA-B-C-D-F 5个蛋白；位于oppA和oppB的间隔区和oppF之后的非编码区各有一个茎环结构，推测分别为oppA和opp基因簇的转录终止子。以细菌OppA的保守结构域SBP_bac_5构建系统发生树，结果显示E.tarda LSE40与同属细菌E.ictaluri的亲缘关系最近，与肠杆菌科细菌的亲缘关系较近，与革兰氏阳性细菌的亲缘关系较远，表明OppA的SBP_bac_5结构域可作为细菌分类鉴定的依据。从E.tarda LSE40 fosmid文库克隆aroA基因全序列，该序列全长1 287bp，编码428个氨基酸，与鲶鱼爱德华氏菌(E. ictaluri)氨基酸相似性在94%，与其他肠杆菌科菌如Escherichia coli和Yersinia enterocolitica相似性在73%-74%。通过In-frame deletion构建了E.tarda LSE40 aroA缺失突变株。与野生型相比，aroA突变株的半数致死量LD50提高了62倍。在牙鲆接种~106cfu/ml的E.tarda细菌时，接种野生型细菌的牙鲆在6天内全部死亡，濒死鱼的细菌数达7.97×108cfu/ 100mg；而接种aroA突变株的牙鲆没有出现死亡，28天后检测不到细菌的存在。实验结果为进一步评价aroA突变株作为减毒活疫苗打下了基础。
其他摘要	The genomic fosmid library of Edwardsiella tarda LSE40 strain was constructed in this study. The obtained library contained approximate 2 500 clones in which the average size of insertion DNA was 33.6 kb. The total capacity of this library was about 84 Mb, which covered more than 16 times of the genome of E.tarda LSE40 (estimation of 5Mb). About 1 000 clones were selected randomly from this fosmid library to perform end-pairing sequencing. A total of 1 741 high-quality sequences were obtained, and each had the average length of 546 bp. The sequences are sum up to 949 997bp, which covered about 19% of the E.tarda genome. KO(KEGG Orthology) annotations were acquired by the KEGG automatic server KAAS for analysis of the metabolic pathway of the sequences. Results indicated that there were 932, 283, 220, 64 and 16 sequences related to pathway of Metabolism, Environmental Information Processing, Genetic Information Processing, Cellular Processing and Human Diseases, respectively. Meanwhile, a total of 61 sequences were found to be virulence-related by BlastX, which closely corresponds to results in the “Common Themes in Microbial Pathogenicity”. The Oligopeptide Permease (opp) gene cluster was screened and cloned from the fosmid library of E.tarda. The obtained opp gene cluster spanned 6 741bp which contains five ORFs encoding five putative proteins, OppA, OppB, OppC, OppD and OppF, respectively. Two potential stem-loop structures located in the spacer region between oppA and oppB, and at downstream of oppF, are presumed as the terminators of oppA and opp, respectively. Phylogenetic tree was constructed based on the OppA conserved domain SBP_bac_5. Results showed that E.tarda LSE40 is most closely related to E. ictaluri, closely related to other Enterobacteriaceae bacteria, and less related to Gram positive bacteria, suggesting that the SBP_bac_5 domain of OppA can be used as a molecular marker in the bacterial identification. The full length sequence of E.tarda LSE40 aroA was screened and cloned from the fosmid library. It had a full-length of 1 287bp, encoding 428 amino acids, which showed 94% identity with that of Edwardsiella ictaluri, 73%-74% identity with that of other Enterobacteriaceae bacteria. The in-frame deletion mutant of aroA was constructed. This mutant showed 62 times increase of 50 percent lethal dose (LD50) in comparison to the wild-type strain. All the flounder fish died in 6 days post-injection of ~106 cfu/ml of wild-type bacteria, and the moribund fish showed 7.97×108cfu/100mg bacteria in kidney tissues. While no mortalities were found in fish injected with ~106 cfu/ml of aroA mutant bacteria, and almost no aroA mutant bacteria were re-isolated from the kidney tissue 28 days post-injection. Thus, the aroA mutant will be used in the future study for identification its potential efficacy as a live attenuated vaccine against E. tarda infection.
页数	84
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/1527
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	杨佳银. 迟缓爱德华氏菌基因组fosmid文库的构建、opp基因簇的克隆及aroA基因缺失突变株的构建[D]. 海洋研究所. 中国科学院海洋研究所,2008.

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