军事医学
軍事醫學
군사의학
Military Medical Sciences
2015年
9期
672-676
,共5页
王红朵%刘子中%王子迎%杨瑞馥%韩延平
王紅朵%劉子中%王子迎%楊瑞馥%韓延平
왕홍타%류자중%왕자영%양서복%한연평
鼠疫耶尔森菌%pPCP1质粒%小RNA%生物膜%毒力
鼠疫耶爾森菌%pPCP1質粒%小RNA%生物膜%毒力
서역야이삼균%pPCP1질립%소RNA%생물막%독력
Yersinia pestis%pPCP1%sRNA%biofilm%virulence
目的：鉴定鼠疫耶尔森菌外源获得性pPCP1质粒编码的小RNA，并评价其在生物膜、压力耐受性和毒力方面的功能。方法基于前期鼠疫菌转录组测序结果，分析获得外源性pPCP1质粒编码的高丰度小RNA，提取鼠疫菌总RNA，利用Northern印迹实验验证这些小RNA存在与否。利用λ-Red同源重组技术敲除pPCP1质粒上已证实的小RNA，并对小RNA缺失株在生物膜褶皱形成、高盐耐受性和致病性等方面进行初步的表型筛选。结果与结论 Northern印迹法鉴定了鼠疫菌pPCP1质粒上7个小RNA的存在。在此基础上，获得5个小RNA的缺失突变株，其中sR3446缺失后鼠疫菌生物膜褶皱形成能力减弱；sR3446、sR3457、sR4338和sR4340缺失后鼠疫菌对高盐环境的耐受能力减弱，而sR6143缺失后对高盐环境的耐受能力增强；sR4338和sR4340缺失后鼠疫菌毒力有所减弱。上述结果提示，鼠疫菌外源获得性pPCP1质粒上的小RNA在鼠疫菌应激和致病过程中可能发挥重要作用。
目的：鑒定鼠疫耶爾森菌外源穫得性pPCP1質粒編碼的小RNA，併評價其在生物膜、壓力耐受性和毒力方麵的功能。方法基于前期鼠疫菌轉錄組測序結果，分析穫得外源性pPCP1質粒編碼的高豐度小RNA，提取鼠疫菌總RNA，利用Northern印跡實驗驗證這些小RNA存在與否。利用λ-Red同源重組技術敲除pPCP1質粒上已證實的小RNA，併對小RNA缺失株在生物膜褶皺形成、高鹽耐受性和緻病性等方麵進行初步的錶型篩選。結果與結論 Northern印跡法鑒定瞭鼠疫菌pPCP1質粒上7箇小RNA的存在。在此基礎上，穫得5箇小RNA的缺失突變株，其中sR3446缺失後鼠疫菌生物膜褶皺形成能力減弱；sR3446、sR3457、sR4338和sR4340缺失後鼠疫菌對高鹽環境的耐受能力減弱，而sR6143缺失後對高鹽環境的耐受能力增彊；sR4338和sR4340缺失後鼠疫菌毒力有所減弱。上述結果提示，鼠疫菌外源穫得性pPCP1質粒上的小RNA在鼠疫菌應激和緻病過程中可能髮揮重要作用。
목적：감정서역야이삼균외원획득성pPCP1질립편마적소RNA，병평개기재생물막、압력내수성화독력방면적공능。방법기우전기서역균전록조측서결과，분석획득외원성pPCP1질립편마적고봉도소RNA，제취서역균총RNA，이용Northern인적실험험증저사소RNA존재여부。이용λ-Red동원중조기술고제pPCP1질립상이증실적소RNA，병대소RNA결실주재생물막습추형성、고염내수성화치병성등방면진행초보적표형사선。결과여결론 Northern인적법감정료서역균pPCP1질립상7개소RNA적존재。재차기출상，획득5개소RNA적결실돌변주，기중sR3446결실후서역균생물막습추형성능력감약；sR3446、sR3457、sR4338화sR4340결실후서역균대고염배경적내수능력감약，이sR6143결실후대고염배경적내수능력증강；sR4338화sR4340결실후서역균독력유소감약。상술결과제시，서역균외원획득성pPCP1질립상적소RNA재서역균응격화치병과정중가능발휘중요작용。
Objective To identify small non-coding RNAs encoded by plasmid pPCP1 and investigate their roles in biofilm formation, stress tolerance and/or virulence in Yersinia pestis.Methods Seven plasmid pPCP1-encoded sRNAs were identified by RNA-seq results in Y.pestis in our previous studies.Northern blot was used to validate the presence of the seven sRNAs.The sRNA-deletion mutants were constructed via λ-Red homologous recombination system.The biofilm formation, high salt tolerance and virulence of the phenotypes were compared between Y.pestis WT strain and sRNA mutants.Results and Conclusion The expression of seven pPCP1-encoded sRNAs was validated and the transcript length detected by Northern blotting corresponded to the length observed by RNA-seq.On this basis, five sRNA-deletion mutants were obtained.The capacity of biofilm formation was weakened upon deletion of sR3446.The tolerance of sR3446, sR3457, sR4338 and sR4340 mutants was found weakened in vitro compared to that of wild-type strain,but the tolerance of sR6143 was found increased.Slight virulence attenuation was found in two sRNA mutants ( sR4338 and sR4340 ) .The results suggest that pPCP1-deriving sRNA might be implicated in stress response, biofilm and virulence in Y.pestis.
