本文主要研究内容
作者张贝贝（2019）在《RK21_RS10315毒力基因在变形假单胞菌感染斜带石斑鱼过程中的功能》一文中研究指出：变形假单胞菌(Pseudomonas plecoglossicida)是一种分布广泛的革兰氏阴性菌,并且具有很强的致病性,能够对养殖产业造成严重的经济损失。然而,关于变形假单胞菌致病机制的研究还处于起步阶段,已经验证的毒力基因也非常之少。本研究以变形假单胞菌为病原,以斜带石斑鱼(Epinephelus coioides)为宿主,结合RNA干扰和dual RNA-seq技术研究变形假单胞菌的一个转录调节子基因(RK21_RS10315)在病原-宿主互作中的功能。首先通过比较体外培养变形假单胞菌与被感染24h,48h,72h,96h的斜带石斑鱼脾脏中变形假单胞菌转录组数据的差异挑选出在体内显著高表达的RK21_RS10315基因。针对该基因设计了5个RNAi靶点的shRNA序列,用pCM130/tac质粒分别构建稳定沉默突变株。利用实时荧光定量PCR技术检测沉默效率,结果发现针对574靶点shRNA的沉默效果最好,沉默效率达到了96.1%。然后,分别用沉默株和野生株变形假单胞菌感染斜带石斑鱼,发现野生株感染的斜带石斑鱼体表无明显特征,脾脏表面有密集的大小不等的白色结节,而由于目的基因的沉默导致被感染鱼的死亡率下降了90%,而且脾脏器官的白色结节症状也有所减轻,这也证实了RK21_RS10315毒力候选基因的致病性。将分别被变形假单胞菌野生株和RK21_RS10315-RNAi突变株感染48h的斜带石斑鱼脾脏进行dual RNA-seq,通过两组数据的比较来分析RK21_RS10315基因在变形假单胞菌与斜带石斑鱼的病原-宿主互作中的功能。被感染的斜带石斑鱼脾脏中包含有两部分转录组数据,分别是变形假单胞菌的转录组数据和斜带石斑鱼的转录组数据。分析发现斜带石斑鱼的转录组数据中共有差异基因22010个,其中差异上调的有4321个,差异下调的有17689个,变形假单胞菌的转录组数据中共有差异基因65个,其中差异上调的有50个,差异下调的有15个。斜带石斑鱼的转录组数据中的差异基因通过KEGG富集分析,发现了16个免疫显著相关的通路,其中ko04672和ko04670两个富集通路占有一定的比重。ko04672和ko04670两个富集通路中的19个差异基因(euk-DEMs)与变形假单胞菌转录组数据中的19个差异基因(pro-DEMs)做基因共表达网络分析,结果发现它们之间具有不同程度的相关性,还发现来自变形假单胞菌转录组数据中的ipxA,grpE,yhbJ,truD和suhB这些差异基因在共表达网络中占有重要位置。它们的表达很可能与变形假单胞菌中的RK21_RS10315基因的沉默相关。RK21_RS10315基因影响着变形假单胞菌的致病性,dual RNA-seq同时检测分别被变形假单胞菌野生株和RK21_RS10315-RNAi突变株感染48h的斜带石斑鱼脾脏,转录组数据做进一步的生物信息学分析发现病原宿主差异基因之间的相关性,基因沉默后的病原-宿主互作证实了与变形假单胞菌中的RK21_RS10315基因密切相关。
Abstract
bian xing jia chan bao jun (Pseudomonas plecoglossicida)shi yi chong fen bu an fan de ge lan shi yin xing jun ,bing ju ju you hen jiang de zhi bing xing ,neng gou dui yang shi chan ye zao cheng yan chong de jing ji sun shi 。ran er ,guan yu bian xing jia chan bao jun zhi bing ji zhi de yan jiu hai chu yu qi bu jie duan ,yi jing yan zheng de du li ji yin ye fei chang zhi shao 。ben yan jiu yi bian xing jia chan bao jun wei bing yuan ,yi xie dai dan ban yu (Epinephelus coioides)wei su zhu ,jie ge RNAgan rao he dual RNA-seqji shu yan jiu bian xing jia chan bao jun de yi ge zhuai lu diao jie zi ji yin (RK21_RS10315)zai bing yuan -su zhu hu zuo zhong de gong neng 。shou xian tong guo bi jiao ti wai pei yang bian xing jia chan bao jun yu bei gan ran 24h,48h,72h,96hde xie dai dan ban yu pi zang zhong bian xing jia chan bao jun zhuai lu zu shu ju de cha yi tiao shua chu zai ti nei xian zhe gao biao da de RK21_RS10315ji yin 。zhen dui gai ji yin she ji le 5ge RNAiba dian de shRNAxu lie ,yong pCM130/taczhi li fen bie gou jian wen ding chen mo tu bian zhu 。li yong shi shi ying guang ding liang PCRji shu jian ce chen mo xiao lv ,jie guo fa xian zhen dui 574ba dian shRNAde chen mo xiao guo zui hao ,chen mo xiao lv da dao le 96.1%。ran hou ,fen bie yong chen mo zhu he ye sheng zhu bian xing jia chan bao jun gan ran xie dai dan ban yu ,fa xian ye sheng zhu gan ran de xie dai dan ban yu ti biao mo ming xian te zheng ,pi zang biao mian you mi ji de da xiao bu deng de bai se jie jie ,er you yu mu de ji yin de chen mo dao zhi bei gan ran yu de si wang lv xia jiang le 90%,er ju pi zang qi guan de bai se jie jie zheng zhuang ye you suo jian qing ,zhe ye zheng shi le RK21_RS10315du li hou shua ji yin de zhi bing xing 。jiang fen bie bei bian xing jia chan bao jun ye sheng zhu he RK21_RS10315-RNAitu bian zhu gan ran 48hde xie dai dan ban yu pi zang jin hang dual RNA-seq,tong guo liang zu shu ju de bi jiao lai fen xi RK21_RS10315ji yin zai bian xing jia chan bao jun yu xie dai dan ban yu de bing yuan -su zhu hu zuo zhong de gong neng 。bei gan ran de xie dai dan ban yu pi zang zhong bao han you liang bu fen zhuai lu zu shu ju ,fen bie shi bian xing jia chan bao jun de zhuai lu zu shu ju he xie dai dan ban yu de zhuai lu zu shu ju 。fen xi fa xian xie dai dan ban yu de zhuai lu zu shu ju zhong gong you cha yi ji yin 22010ge ,ji zhong cha yi shang diao de you 4321ge ,cha yi xia diao de you 17689ge ,bian xing jia chan bao jun de zhuai lu zu shu ju zhong gong you cha yi ji yin 65ge ,ji zhong cha yi shang diao de you 50ge ,cha yi xia diao de you 15ge 。xie dai dan ban yu de zhuai lu zu shu ju zhong de cha yi ji yin tong guo KEGGfu ji fen xi ,fa xian le 16ge mian yi xian zhe xiang guan de tong lu ,ji zhong ko04672he ko04670liang ge fu ji tong lu zhan you yi ding de bi chong 。ko04672he ko04670liang ge fu ji tong lu zhong de 19ge cha yi ji yin (euk-DEMs)yu bian xing jia chan bao jun zhuai lu zu shu ju zhong de 19ge cha yi ji yin (pro-DEMs)zuo ji yin gong biao da wang lao fen xi ,jie guo fa xian ta men zhi jian ju you bu tong cheng du de xiang guan xing ,hai fa xian lai zi bian xing jia chan bao jun zhuai lu zu shu ju zhong de ipxA,grpE,yhbJ,truDhe suhBzhe xie cha yi ji yin zai gong biao da wang lao zhong zhan you chong yao wei zhi 。ta men de biao da hen ke neng yu bian xing jia chan bao jun zhong de RK21_RS10315ji yin de chen mo xiang guan 。RK21_RS10315ji yin ying xiang zhao bian xing jia chan bao jun de zhi bing xing ,dual RNA-seqtong shi jian ce fen bie bei bian xing jia chan bao jun ye sheng zhu he RK21_RS10315-RNAitu bian zhu gan ran 48hde xie dai dan ban yu pi zang ,zhuai lu zu shu ju zuo jin yi bu de sheng wu xin xi xue fen xi fa xian bing yuan su zhu cha yi ji yin zhi jian de xiang guan xing ,ji yin chen mo hou de bing yuan -su zhu hu zuo zheng shi le yu bian xing jia chan bao jun zhong de RK21_RS10315ji yin mi qie xiang guan 。
论文参考文献
论文详细介绍
论文作者分别是来自集美大学的张贝贝，发表于刊物集美大学2019-07-12论文，是一篇关于水产病害论文,变形假单胞菌论文,毒力基因论文,病原宿主互作论文,集美大学2019-07-12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，资料来自集美大学2019-07-12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：水产病害论文; 变形假单胞菌论文; 毒力基因论文; 病原宿主互作论文; 集美大学2019-07-12论文;