| 1 | 溶藻弧菌T3SS exsD基因敲除突变株构建及其表型特征显示文摘【目的】构建溶藻弧菌(Vibrio alginolyticus)Ⅲ型分泌系统(Type Ⅲ secretion system,T3SS)中的调控蛋白操纵子ExsD基因缺失株,研究exsD缺失株表型特征。【方法】采用overlapPCR技术构建缺失株ΔexsD,PCR检测缺失株ΔexsD的遗传稳定性,比较缺失株与野生株之间的生长速度、泳动能力、胞外酶活性、药物敏感性、毒力,分别采用结晶紫和共聚焦电镜方法测定生物膜的差异变化,通过qRT-PCR方法分析exsD的缺失对T3SS效应蛋白Hop转录的影响。【结果】缺失株ΔexsD构建成功;与野生株相比,ΔexsD遗传稳定性和生长速度无显著差别,但泳动能力极显著上升(P <0.01),胞外蛋白酶活性显著上升(P <0.05),形成生物膜能力在24 h时提高(P <0.05);相比野生株,ΔexsD对米诺环素、庆大霉素、卡那霉素、多西环素、新霉素的敏感性从耐药变成中度敏感;ΔexsD缺失株的毒力显著上升,野生株对斑马鱼的半数致死剂量是缺失株的3.68倍。实时荧光定量结果显示,与HY9901野生型相比,exsD基因的缺失增加了效应蛋白Hop的转录表达。【结论】exsD基因对T3SS的致病性起负调控作用,为进一步阐明exsD基因在溶藻弧菌中的作用机理提供一定的理论基础。 | 王俊霖 招茵 苏茵茵 周诗慧 曾福源 谢妙 王娜 简纪常 庞欢瑛 | 2021 | 广东海洋大学学报2021，41，5： | 6 |
| 2 | Cloning and Bioinformatics Analysis of TpiA Gene of Vibrio alginolyticus HY9901显示文摘[Objectives]To clone and analyze the TpiA gene of Vibrio alginolyticus HY9901.[Methods]According to the TpiA gene sequence of V.alginolyticus,a pair of specific primers was designed,and its full length was amplified by PCR.[Results]The full length of TpiA gene is 771 bp,encoding 256 amino acid residues in total,and the NCBI accession number is OM906798.According to the deduced amino acid sequence,its molecular weight was predicted to be about 26.97548 kDa,and its isoelectric point was 4.78.The amino acid sequence of the N-terminal signal peptide structure was predicted,and it was found that there was no obvious signal peptide cleavage site,no signal peptide,and no transmembrane region;the amino acid sequence contained 3 N-glycosylation sites,4 protein kinase C phosphorylation sites,2 casein kinase II phosphorylation sites,6 N-myristoylation sites,7 microbody C-terminal target signal site,and 1 triose phosphate isomerase active site.The prediction results of protein subcellular localization showed that TpiA may be located in mitochondria or cytoplasm,with probability of 39.1%and 34.8%,respectively.The amino acid sequence of the TpiA gene of V.alginolyticus shared 98.83%-99.61%homology with other Vibrio species,and it was clustered into the same subfamily with Vibrio parahaemolyticus and had a close relationship.In the secondary structure prediction,the proportions ofα-helix,random coil and extended chain were 44.53%,41.41%and 14.06%,respectively,and the similarity of its tertiary structure model to template 1aw1.1.A was 85.16%.[Conclusions]This study is intended to provide a basis for further research on the role of TpiA gene in the type III secretion system and related research on antibiotic resistance. | Ling ZUO Liangchuan CHEN Shuai YANG Xing XIAO Fuyuan ZENG Junlin WANG Weijie ZHANG Huanying PANG | 2022 | Asian Agricultural Research2022，14，5： | 0 |
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