哈维氏弧菌运动相关基因功能研究
摘要
弧菌病(Vibriosis)是一种全球范围内发生的细菌性疾病。其中哈维氏弧菌(Vibrio harveyi)是一种水产养殖业中非常重要的致病菌,严重威胁到水产养殖动物的健康,给海水鱼类、虾类及其他养殖对象造成巨大经济损失。因此,有关哈维氏弧菌致病机理的研究在国内外受到高度重视。现有研究发现哈维氏弧菌致病机理与胞外产物、溶血素等均相关,但有关其运动性及运动在细菌感染早期的作用尚未见报道。鉴于运动在细菌致病早期的重要作用,本研究以哈维氏弧菌为研究对象,寻找运动相关基因,试图揭示运动相关基因的功能,初步了解运动性与致病性之间的相互联系,以期对哈维氏弧菌乃至其它弧菌致病机制和防治研究提供一定的理论依据。
本研究将从Escherichia coli Sm10(pLOFKm)中携带的转座子mini-Tn10的自杀性质粒pLOFKm引入受体菌哈维氏弧菌TS-628菌株中,构建突变株库。我们从大约3000个转座突变株中筛选到3株运动缺陷型菌株。避开转座子内部酶切位点选用XbaI酶切突变株的基因组DNA,在连接酶的作用下使基因组DNA的酶切片段环化,以这些环化的片段为模板,根据转座子序列设计引物进行反向PCR,将反向PCR产物克隆到T载体测序,获得转座子侧翼未知基因的部分序列,根据获得的序列在基因库中寻找同源基因,结果发现这465bp的侧翼序列表现出与哈维氏弧菌ATCC BAA-1116菌株基因组3612978至3613443序列具有很高的同源性(96.9%),该序列在哈维氏弧菌ATCC BAA-1116菌株中为一个未知功能的蛋白,此外,该序列还与副溶血弧菌(V.parahaemolyticus)RIMD 2210633菌株基因组中的pgk (磷酸甘油酸激酶)基因具有第二高(91.8%)的同源性。突变株H2反向PCR产物片段长约950bp,其中356bp为获得的插入位点侧翼的未知基因序列,该序列经过同源性比较发现与哈维氏弧菌ATCC BAA-1116菌株基因组3203663至3204019序列的同源性最高(94.6%),与溶藻弧菌(V.alginolyticus)HY9901菌株鞭毛蛋白基因flaB具有第二高的同源性(92.4%)。
最后鉴定了突变株在生长、趋化、早期黏附及生物膜形成等方面的生物学特性。结果发现,突变株H1生长参数明显不同于野生株。趋化性测定显示,3株突变株均都趋化性减弱。对野生型和3株突变型菌株早期粘附研究发现,野生型菌株粘附能力均强于3株突变株。对于生物膜形成研究说明野生型菌株形成生物膜的能力强于突变株,而在3株突变株中H2型菌株形成的生物膜的能力最弱。
总之,通过本研究寻找到哈维氏弧菌2个运动相关基因的部分序列,对这两段序列进行同源性分析及基因功能研究,结果认为这两个片段的基因功能为运动相关基因。它们的功能分别与pgk(磷酸甘油酸激酶)基因和鞭毛蛋白基因flaB相类似。
Vibriosis is a kind of bacteriosis widely spreading on the earth.Vibrio harveyi is a vital pathogen in aquaculture,which is severely harmful to wild fish、custacea、shellfish and other animals of aquaculture.Therefore,the pathogenic mechanism of V.harveyi was paid much attention in recent years.The present research showed that the pathogenic mechanism of V.harveyi was related to ECP(Extracellular Product)、hemolysin and so on,while there has been not any report on motility and the role of motility on the early stage of infection of this bacterium.As the important role of motility in infection of bacterial,motile-related genes of V. harveyi was identified and function of these genes were characterized in the present study,in order to reveal the relationship between motility and pathogenicity.we wish it could provide us Rationale of Pathogenicity and Prevention of V.harveyi.The results of this study provided further understanding of the pathogenic mechanism of V.harveyi.
The suicide plasmid pLOFKm,carring a transposon mini-Tn10,was transferred to the recipient V.harveyi to generate an insertional mutant library by cell conjugation between the donor Escherichia coli Sm10 (pLOFKm) and the recipient V.harveyi TS-628 by filter mating.More than tow thousand individual insertion transposon mutants were screened and three mutants exhibited defective in motility.465bp sequence of mutant H1 flanking transposon showed the highest identity (96.9%) to a hypothetical protein gene of V.harveyi ATCC BAA-1116 at genome position 3612978 to 3613443 and the second-highest identity (91.8%) to the pgk gene of V.parahaemolyticus RIMD 2210633.To the H2,356bp flanking sequence showed the highest identity (94.6%) to a hypothetical protein gene of V.harveyi ATCC BAA-1116 at genome position 3203663 to 3204019 and the second-highest identity (92.4%) to the flaB gene of V.alginolyticus HY9901.
The biological characteristics of the mutants including growth、chemotactic、early stage of adhesion、biofilm formation were determined . The results displayed that the growth characteristics of H1 are abviously different from the wild type strain and the growth parameters of H2 and H3 were similar with those of the wild type strain.Chemotactic testing showed that all the three mutants were greatly impaired in chemotaxis.The study on the early stage of adhesion of the wild type strain and the three mutants reveal that the wild type strain exhibited the strongest adhesion ability and all the mutants exhibited weaker adhesion ability.The biofilm formation assay revealed that the wild type strain produced the greatest biofilm and the mutant H2 produced the minimal biofilm.
Hence, the target gene in H1 and H2 were first identified to be the motile-related genes in V. harveyi in this study and their function is similar with the pgk gene and flaB gene respectively.
本研究将从Escherichia coli Sm10(pLOFKm)中携带的转座子mini-Tn10的自杀性质粒pLOFKm引入受体菌哈维氏弧菌TS-628菌株中,构建突变株库。我们从大约3000个转座突变株中筛选到3株运动缺陷型菌株。避开转座子内部酶切位点选用XbaI酶切突变株的基因组DNA,在连接酶的作用下使基因组DNA的酶切片段环化,以这些环化的片段为模板,根据转座子序列设计引物进行反向PCR,将反向PCR产物克隆到T载体测序,获得转座子侧翼未知基因的部分序列,根据获得的序列在基因库中寻找同源基因,结果发现这465bp的侧翼序列表现出与哈维氏弧菌ATCC BAA-1116菌株基因组3612978至3613443序列具有很高的同源性(96.9%),该序列在哈维氏弧菌ATCC BAA-1116菌株中为一个未知功能的蛋白,此外,该序列还与副溶血弧菌(V.parahaemolyticus)RIMD 2210633菌株基因组中的pgk (磷酸甘油酸激酶)基因具有第二高(91.8%)的同源性。突变株H2反向PCR产物片段长约950bp,其中356bp为获得的插入位点侧翼的未知基因序列,该序列经过同源性比较发现与哈维氏弧菌ATCC BAA-1116菌株基因组3203663至3204019序列的同源性最高(94.6%),与溶藻弧菌(V.alginolyticus)HY9901菌株鞭毛蛋白基因flaB具有第二高的同源性(92.4%)。
最后鉴定了突变株在生长、趋化、早期黏附及生物膜形成等方面的生物学特性。结果发现,突变株H1生长参数明显不同于野生株。趋化性测定显示,3株突变株均都趋化性减弱。对野生型和3株突变型菌株早期粘附研究发现,野生型菌株粘附能力均强于3株突变株。对于生物膜形成研究说明野生型菌株形成生物膜的能力强于突变株,而在3株突变株中H2型菌株形成的生物膜的能力最弱。
总之,通过本研究寻找到哈维氏弧菌2个运动相关基因的部分序列,对这两段序列进行同源性分析及基因功能研究,结果认为这两个片段的基因功能为运动相关基因。它们的功能分别与pgk(磷酸甘油酸激酶)基因和鞭毛蛋白基因flaB相类似。
Vibriosis is a kind of bacteriosis widely spreading on the earth.Vibrio harveyi is a vital pathogen in aquaculture,which is severely harmful to wild fish、custacea、shellfish and other animals of aquaculture.Therefore,the pathogenic mechanism of V.harveyi was paid much attention in recent years.The present research showed that the pathogenic mechanism of V.harveyi was related to ECP(Extracellular Product)、hemolysin and so on,while there has been not any report on motility and the role of motility on the early stage of infection of this bacterium.As the important role of motility in infection of bacterial,motile-related genes of V. harveyi was identified and function of these genes were characterized in the present study,in order to reveal the relationship between motility and pathogenicity.we wish it could provide us Rationale of Pathogenicity and Prevention of V.harveyi.The results of this study provided further understanding of the pathogenic mechanism of V.harveyi.
The suicide plasmid pLOFKm,carring a transposon mini-Tn10,was transferred to the recipient V.harveyi to generate an insertional mutant library by cell conjugation between the donor Escherichia coli Sm10 (pLOFKm) and the recipient V.harveyi TS-628 by filter mating.More than tow thousand individual insertion transposon mutants were screened and three mutants exhibited defective in motility.465bp sequence of mutant H1 flanking transposon showed the highest identity (96.9%) to a hypothetical protein gene of V.harveyi ATCC BAA-1116 at genome position 3612978 to 3613443 and the second-highest identity (91.8%) to the pgk gene of V.parahaemolyticus RIMD 2210633.To the H2,356bp flanking sequence showed the highest identity (94.6%) to a hypothetical protein gene of V.harveyi ATCC BAA-1116 at genome position 3203663 to 3204019 and the second-highest identity (92.4%) to the flaB gene of V.alginolyticus HY9901.
The biological characteristics of the mutants including growth、chemotactic、early stage of adhesion、biofilm formation were determined . The results displayed that the growth characteristics of H1 are abviously different from the wild type strain and the growth parameters of H2 and H3 were similar with those of the wild type strain.Chemotactic testing showed that all the three mutants were greatly impaired in chemotaxis.The study on the early stage of adhesion of the wild type strain and the three mutants reveal that the wild type strain exhibited the strongest adhesion ability and all the mutants exhibited weaker adhesion ability.The biofilm formation assay revealed that the wild type strain produced the greatest biofilm and the mutant H2 produced the minimal biofilm.
Hence, the target gene in H1 and H2 were first identified to be the motile-related genes in V. harveyi in this study and their function is similar with the pgk gene and flaB gene respectively.
引文
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