本文主要研究内容
作者唐蓉（2019）在《鱼源致腐菌荧光假单胞菌群体感应LuxI/LuxR鉴定及生理功能研究》一文中研究指出：荧光假单胞菌(Pseudomonas fluorescens)具有较高的蛋白质、脂质水解力,和良好的嗜冷性,是生鱼、肉类和乳制品中重要的革兰氏阴性腐败菌。群体感应是一种细胞间的交流机制,参与毒力因子分泌、生物被膜形成、食品腐败等的调控。现有研究表明希瓦氏菌、气单胞菌、假单胞菌等革兰氏阴性的致腐性与群体感应密切相关,然而,,P.fluuoescens中AHLs介导的QS系统分子信息及其调控生物被膜、腐败和抗胁迫性的机制的研究较少。鉴于此,本研究以冷藏腐败大黄鱼中分离鉴定的优势腐败菌P.fluorescensPF07为研究对象,采用生物报菌法和高效液相色谱串联质谱(LC-MS/MS)检测PF07中的AHLs活性,利用基因框架测序技术预测PF07全基因组,并对获得的LuxI和LuxR蛋白进行生物信息学分析;构建并验证luxl和luxR基因缺失株,比较研究野生株和缺失株的生长、生物被膜、致腐表型及抗胁迫性差异变化,评价luxI基因缺失株中外源添加C4-HSL的影响;基于转录组学测序技术研究luxI和luxR基因缺失对P.fluorescens的基因表达及代谢通路的影响,旨在更加全面地了解致腐菌LuxI和LuxR系统,为研发新型食品保鲜剂提供理论支持。主要结果如下:研究发现PfuorescensPF07上清液中含有C4-HSL、C10-HSL和C14-HSL三种信号分子,且C4-HSL含量最高。PF07基因框架测序显示,预测全基因组大小约为6.59Mb,编码基因约5573个,长度约为5,352,570bp。PF07中预测出的ncRNA 有 3 种(tRNA,rRNA 和 sRNA),Prophage 有 6 个,CRISPRs 有 70 个,次级代谢产物基因簇6种,其中hserlactone(homoserine lactone)是与群体感应相关的基因簇。采用COG聚类分析PF07功能时,从Signal transduction mechanism类型的基因中发现群体感应相关的合成酶GM004424(LuxI)和受体蛋白GM004425(LuxR)。两个蛋白的生物信息学分析发现,PF07中LuxI/LuxR与荧光假单胞菌PFuk4中同源蛋白具有高度的同一性和相似性,并且与其他菌株中六种LuxI型合成酶和LuxR受体蛋白具有相同的保守氨基酸和相似的蛋白质3D结构,因此认为PF07中LuxI/LuxR是一对同源的群体感应合成酶和受体蛋白。构建了P.fuorescensPF07luxl和luxR基因缺失株,比较分析野生株及两株突变株△luxI/△luuxR在生物被膜形成、致腐能力和抗胁迫性间的差异,并评价C4-HSL添加对△luxI的影响。结果发现,缺失株△luxI中C4-HSL几乎散失,△luxR中降低约38.60%,但对C10-HSL和C14-HSL活性无影响。在30℃和4℃下luxI和luxR基因缺失对细菌生长无影响,分别在18 h和144 h达到稳定期。luxI和luxR基因缺失都导致生物被膜的形成减弱,且降低细菌的粘附能力及胞外多糖形成量,却促进细菌泳动性。△luxI和△luxR在30℃下被膜结晶紫量分别降低17.22%和14.56%,而4℃分别减少27.51%和22.48%;C4-HSL部分回补被膜含量,30℃和4℃下最高分别回复12.82%和23.66%。CLSM显示两种温度下△luxI和△luxR形成的被膜都更薄且结构较疏松,SEM观察到AluxI和AluxR胞外分泌物减少。luxI和luxR基因缺失也降低菌体胞外蛋白酶和嗜铁素的产生,特别是蛋白酶活性,最高时分别降低了 11.94%和12.51%,但不影响TVB-N含量。外源添加C4-HSL可部分恢复△luxl中嗜铁素和蛋白酶的缺陷,其中对蛋白酶作用较弱,最高仅能恢复 9.2%。此外Aluxl和AluxR在20mM H2O2、30%(m/v)NaCl、50℃和 150μg/mL结晶紫中的抗胁迫力弱于野生株,其中H2O2处理三者间差异最大,野生株的存活率分别是△luxR和AluxI的15.31倍和18.71倍。转录组学分析显示,P.fuorescensPF07中LuxI/LuxR敲除会影响多种生物被膜相关的基因,包括下调多糖相关的基因、上调pst系统相关的基因,AluxR中还上调PhoB/U双组分系统。而对pst系统与PhoB/U双组分系统基因上调,可能导致下调细胞内c-di-GMP含量,从而促进细胞的泳动性。同时,两个缺失株△luxI和△luxR中参与胞外蛋白酶产生(aprX),嗜铁素生物合成(ornithine monooxygenase)和应激反应(rpos、OmsC、HslV)基因的下调。还有在△luxR和△luxI中其他与腐败相关基因的表达水平也下调,如脂质代谢,硫化合物代谢,芳香族化合物和氨基酸代谢,异味产生等;LuxR在△luxI中的表达水平下调。外源添加C4-HSL仅恢复AluxI的部分基因,如与嗜铁素合成相关的基因ornithine monooxygenase、与多糖合成相关的基因 polysaccharide biosynthesis protein,与绿脓素产生相关基因pyocinR2holin,与应激反应相关基因osmC,hslV等。采用荧光定量PCR,研究验证aprX、fihA、luxI、luxR、orm、pbp和rpoS基因在四组菌中表达量的变化,其结果与转录组测序基本一致,且都能映证表型变化的结果,证明转录组测序结果较为可靠。研究表明,LuxI/LuxR是荧光假单胞菌PF07中一对同源的群体感应合成酶和受体蛋白,且LuxI/LuxR能通过上调多糖相关的基因、下调pst系统相关基因及PhoB/U双组分系统基因来促进生物被膜的形成,上调胞外蛋白、嗜铁素、脂质代谢等腐败相关基因来促进腐败,以及上调应激反应相关基因来提高其抗胁迫性。从生理表型和基因表达差异变化,证实了群体感应LuxI/LuxR对荧光假单胞菌被膜形成、致腐能力以及抗胁迫性的正向调节作用。
Abstract
ying guang jia chan bao jun (Pseudomonas fluorescens)ju you jiao gao de dan bai zhi 、zhi zhi shui jie li ,he liang hao de shi leng xing ,shi sheng yu 、rou lei he ru zhi pin zhong chong yao de ge lan shi yin xing fu bai jun 。qun ti gan ying shi yi chong xi bao jian de jiao liu ji zhi ,can yu du li yin zi fen bi 、sheng wu bei mo xing cheng 、shi pin fu bai deng de diao kong 。xian you yan jiu biao ming xi wa shi jun 、qi chan bao jun 、jia chan bao jun deng ge lan shi yin xing de zhi fu xing yu qun ti gan ying mi qie xiang guan ,ran er ,,P.fluuoescenszhong AHLsjie dao de QSji tong fen zi xin xi ji ji diao kong sheng wu bei mo 、fu bai he kang xie pai xing de ji zhi de yan jiu jiao shao 。jian yu ci ,ben yan jiu yi leng cang fu bai da huang yu zhong fen li jian ding de you shi fu bai jun P.fluorescensPF07wei yan jiu dui xiang ,cai yong sheng wu bao jun fa he gao xiao ye xiang se pu chuan lian zhi pu (LC-MS/MS)jian ce PF07zhong de AHLshuo xing ,li yong ji yin kuang jia ce xu ji shu yu ce PF07quan ji yin zu ,bing dui huo de de LuxIhe LuxRdan bai jin hang sheng wu xin xi xue fen xi ;gou jian bing yan zheng luxlhe luxRji yin que shi zhu ,bi jiao yan jiu ye sheng zhu he que shi zhu de sheng chang 、sheng wu bei mo 、zhi fu biao xing ji kang xie pai xing cha yi bian hua ,ping jia luxIji yin que shi zhu zhong wai yuan tian jia C4-HSLde ying xiang ;ji yu zhuai lu zu xue ce xu ji shu yan jiu luxIhe luxRji yin que shi dui P.fluorescensde ji yin biao da ji dai xie tong lu de ying xiang ,zhi zai geng jia quan mian de le jie zhi fu jun LuxIhe LuxRji tong ,wei yan fa xin xing shi pin bao xian ji di gong li lun zhi chi 。zhu yao jie guo ru xia :yan jiu fa xian PfuorescensPF07shang qing ye zhong han you C4-HSL、C10-HSLhe C14-HSLsan chong xin hao fen zi ,ju C4-HSLhan liang zui gao 。PF07ji yin kuang jia ce xu xian shi ,yu ce quan ji yin zu da xiao yao wei 6.59Mb,bian ma ji yin yao 5573ge ,chang du yao wei 5,352,570bp。PF07zhong yu ce chu de ncRNA you 3 chong (tRNA,rRNA he sRNA),Prophage you 6 ge ,CRISPRs you 70 ge ,ci ji dai xie chan wu ji yin cu 6chong ,ji zhong hserlactone(homoserine lactone)shi yu qun ti gan ying xiang guan de ji yin cu 。cai yong COGju lei fen xi PF07gong neng shi ,cong Signal transduction mechanismlei xing de ji yin zhong fa xian qun ti gan ying xiang guan de ge cheng mei GM004424(LuxI)he shou ti dan bai GM004425(LuxR)。liang ge dan bai de sheng wu xin xi xue fen xi fa xian ,PF07zhong LuxI/LuxRyu ying guang jia chan bao jun PFuk4zhong tong yuan dan bai ju you gao du de tong yi xing he xiang shi xing ,bing ju yu ji ta jun zhu zhong liu chong LuxIxing ge cheng mei he LuxRshou ti dan bai ju you xiang tong de bao shou an ji suan he xiang shi de dan bai zhi 3Djie gou ,yin ci ren wei PF07zhong LuxI/LuxRshi yi dui tong yuan de qun ti gan ying ge cheng mei he shou ti dan bai 。gou jian le P.fuorescensPF07luxlhe luxRji yin que shi zhu ,bi jiao fen xi ye sheng zhu ji liang zhu tu bian zhu △luxI/△luuxRzai sheng wu bei mo xing cheng 、zhi fu neng li he kang xie pai xing jian de cha yi ,bing ping jia C4-HSLtian jia dui △luxIde ying xiang 。jie guo fa xian ,que shi zhu △luxIzhong C4-HSLji hu san shi ,△luxRzhong jiang di yao 38.60%,dan dui C10-HSLhe C14-HSLhuo xing mo ying xiang 。zai 30℃he 4℃xia luxIhe luxRji yin que shi dui xi jun sheng chang mo ying xiang ,fen bie zai 18 hhe 144 hda dao wen ding ji 。luxIhe luxRji yin que shi dou dao zhi sheng wu bei mo de xing cheng jian ruo ,ju jiang di xi jun de nian fu neng li ji bao wai duo tang xing cheng liang ,que cu jin xi jun yong dong xing 。△luxIhe △luxRzai 30℃xia bei mo jie jing zi liang fen bie jiang di 17.22%he 14.56%,er 4℃fen bie jian shao 27.51%he 22.48%;C4-HSLbu fen hui bu bei mo han liang ,30℃he 4℃xia zui gao fen bie hui fu 12.82%he 23.66%。CLSMxian shi liang chong wen du xia △luxIhe △luxRxing cheng de bei mo dou geng bao ju jie gou jiao shu song ,SEMguan cha dao AluxIhe AluxRbao wai fen bi wu jian shao 。luxIhe luxRji yin que shi ye jiang di jun ti bao wai dan bai mei he shi tie su de chan sheng ,te bie shi dan bai mei huo xing ,zui gao shi fen bie jiang di le 11.94%he 12.51%,dan bu ying xiang TVB-Nhan liang 。wai yuan tian jia C4-HSLke bu fen hui fu △luxlzhong shi tie su he dan bai mei de que xian ,ji zhong dui dan bai mei zuo yong jiao ruo ,zui gao jin neng hui fu 9.2%。ci wai Aluxlhe AluxRzai 20mM H2O2、30%(m/v)NaCl、50℃he 150μg/mLjie jing zi zhong de kang xie pai li ruo yu ye sheng zhu ,ji zhong H2O2chu li san zhe jian cha yi zui da ,ye sheng zhu de cun huo lv fen bie shi △luxRhe AluxIde 15.31bei he 18.71bei 。zhuai lu zu xue fen xi xian shi ,P.fuorescensPF07zhong LuxI/LuxRqiao chu hui ying xiang duo chong sheng wu bei mo xiang guan de ji yin ,bao gua xia diao duo tang xiang guan de ji yin 、shang diao pstji tong xiang guan de ji yin ,AluxRzhong hai shang diao PhoB/Ushuang zu fen ji tong 。er dui pstji tong yu PhoB/Ushuang zu fen ji tong ji yin shang diao ,ke neng dao zhi xia diao xi bao nei c-di-GMPhan liang ,cong er cu jin xi bao de yong dong xing 。tong shi ,liang ge que shi zhu △luxIhe △luxRzhong can yu bao wai dan bai mei chan sheng (aprX),shi tie su sheng wu ge cheng (ornithine monooxygenase)he ying ji fan ying (rpos、OmsC、HslV)ji yin de xia diao 。hai you zai △luxRhe △luxIzhong ji ta yu fu bai xiang guan ji yin de biao da shui ping ye xia diao ,ru zhi zhi dai xie ,liu hua ge wu dai xie ,fang xiang zu hua ge wu he an ji suan dai xie ,yi wei chan sheng deng ;LuxRzai △luxIzhong de biao da shui ping xia diao 。wai yuan tian jia C4-HSLjin hui fu AluxIde bu fen ji yin ,ru yu shi tie su ge cheng xiang guan de ji yin ornithine monooxygenase、yu duo tang ge cheng xiang guan de ji yin polysaccharide biosynthesis protein,yu lu nong su chan sheng xiang guan ji yin pyocinR2holin,yu ying ji fan ying xiang guan ji yin osmC,hslVdeng 。cai yong ying guang ding liang PCR,yan jiu yan zheng aprX、fihA、luxI、luxR、orm、pbphe rpoSji yin zai si zu jun zhong biao da liang de bian hua ,ji jie guo yu zhuai lu zu ce xu ji ben yi zhi ,ju dou neng ying zheng biao xing bian hua de jie guo ,zheng ming zhuai lu zu ce xu jie guo jiao wei ke kao 。yan jiu biao ming ,LuxI/LuxRshi ying guang jia chan bao jun PF07zhong yi dui tong yuan de qun ti gan ying ge cheng mei he shou ti dan bai ,ju LuxI/LuxRneng tong guo shang diao duo tang xiang guan de ji yin 、xia diao pstji tong xiang guan ji yin ji PhoB/Ushuang zu fen ji tong ji yin lai cu jin sheng wu bei mo de xing cheng ,shang diao bao wai dan bai 、shi tie su 、zhi zhi dai xie deng fu bai xiang guan ji yin lai cu jin fu bai ,yi ji shang diao ying ji fan ying xiang guan ji yin lai di gao ji kang xie pai xing 。cong sheng li biao xing he ji yin biao da cha yi bian hua ,zheng shi le qun ti gan ying LuxI/LuxRdui ying guang jia chan bao jun bei mo xing cheng 、zhi fu neng li yi ji kang xie pai xing de zheng xiang diao jie zuo yong 。
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论文作者分别是来自浙江工商大学的唐蓉，发表于刊物浙江工商大学2019-06-06论文，是一篇关于荧光假单胞菌论文,群体感应论文,生物被膜论文,腐败论文,抗胁迫性论文,浙江工商大学2019-06-06论文的文章。本文可供学术参考使用，各位学者可以免费参考阅读下载，文章观点不代表本站观点，资料来自浙江工商大学2019-06-06论文网站，若本站收录的文献无意侵犯了您的著作版权，请联系我们删除。
标签：荧光假单胞菌论文; 群体感应论文; 生物被膜论文; 腐败论文; 抗胁迫性论文; 浙江工商大学2019-06-06论文;