本文主要研究内容
作者刘凯（2019）在《基于多组学技术研究长江刀鲚感染线虫后的免疫适应机制》一文中研究指出：长江刀鲚是长江下游流域最为重要的捕捞和消费对象,是目前长江流域唯一能形成稳定渔汛的江海洄游型鱼类。在水域生态环境恶化及人类捕捞胁迫的双重影响下,长江刀鲚资源日趋枯竭,目前年捕捞量仅约为历史最高纪录的2%。本研究系统调查长江刀鲚线虫感染特征及寄生线虫群落特征;基于简化基因组技术和耳石指纹元素技术筛选出遗传背景相似、洄游履历相近的长江刀鲚实验样本,综合利用转录组和代谢组技术研究长江刀鲚感染异尖线虫后相关免疫基因及代谢产物的变化,揭示与感染异尖线虫相关的免疫信号通路,探讨洄游群体感染异尖线虫后所产生的免疫适应性反应。结合长江刀鲚种群生态学研究,进一步探讨长江刀鲚与寄生异尖线虫间的相互作用机制,研究结果对于后续异尖线虫感染对长江刀鲚种群补充的影响研究具有积极意义。本文主要研究内容如下:系统调查了长江刀鲚感染线虫情况。渔汛期内在长江口至鄱阳湖的7个调查断面上共采集长江刀鲚695尾,从541尾样本中检出线虫7271条,总体感染率为77.84%,平均感染强度为13.44±30.68条/尾,平均感染丰度为10.46±27.63条/尾,不同断面间刀鲚样本线虫感染强度和感染丰度差异显著(p<0.05)。利用分子鉴定方法对随机抽样的1160条线虫的鉴定结果显示,共检出异尖科线虫2属6种,分别为派氏异尖线虫(Anisakis pegreffii)、简单异尖线虫(A.simplex)、内弯宫脂线虫(Hysterothylacium aduncum)、费氏宫脂线虫(H.fabri)、中华宫脂线虫(H.sinens)和厦门宫脂线虫(H.amoyense),其中派氏异尖线虫为优势种,占84.84%。ITS序列分析结果显示,派氏异尖线虫的单倍型多样性(Hd)为0.174,核苷酸多样性(π)为0.00028,各断面群体间没有发生明显的遗传分化(Fst<0.05),总基因流(Nm)为75.74,群体间基因交流充分。研究了长江刀鲚抽样群体遗传背景。利用2b-RAD技术对165尾长江刀鲚样本进行测序,经筛选和聚类分析后,获得373244个标签,其平均Unique标签数为289602个,Unique标签比对率变幅为73.77%-80.25%。利用SOAP软件将过滤后的Enzyme Reads比对到参考序列,以最大似然法(ML)进行SNP标记分型,共获得SNP标记36975个,其中高度多态性的SNP(PIC>0.5)位点共计26个。群体二态性SNP标记36469个,其中以A/G和C/T转换的形式为主,分别有10013个和12881个,分别占SNP总量的27.08%和34.84%;转换与颠换比为1.69。分组间平均观测杂合度Ho变化范围为0.1286-0.1408,平均期望杂合度He最小为0.1340,最大为0.1446,各分组间遗传分化系数均低于0.005。表明长江刀鲚洄游群体遗传分化程度极低,个体信息交流紧密,杂合度较高,具有丰富的遗传多样性。通过本次筛选验证可保证长江刀鲚组学实验样本具有相似的遗传背景,进而排除实验样本种质差异对分析结果的干扰。研究了长江刀鲚抽样群体生境履历。利用耳石指纹元素技术对81尾长江刀鲚样本的生境履历进行反演,共发现3种生态类型:江-海洄游型(91.36%)、江-河口洄游型(6.17%)和淡水定居型(2.47%)。就感染线虫与生境履历的关系而言,所有感染线虫的长江刀鲚样本均具有江海洄游履历,但具有江海洄游履历的长江刀鲚不一定感染线虫,淡水定居型均未感染线虫。本次筛选验证可保证长江刀鲚组学样本具有相近的洄游履历,进而排除实验样本因栖息生境的差异对分析结果产生干扰。进行了长江刀鲚肝脏转录组分析。利用RNA-seq技术对感染异尖线虫后的长江刀鲚肝脏组织进行表达谱分析。测序完成后,对原始数据进行过滤、组装后共获得62604条Unigene。通过GO和KEGG等生物信息学方法对Unigene进行注释和分析,并预测其功能。对基因表达差异分析,共获得961个差异表达基因,包括545个上调基因和416个下调基因。10个宿主基因qRT-PCR检测结果与RNA-seq结果一致,进一步证明RNA测序结果的真实性和可靠性。通过pathway富集分析,筛选出免疫相关基因和免疫信号通路,包括参与抗原加工和呈递过程的MHCⅡ、TCR、CTSL以及CIITA基因和参与T细胞受体信号通路的TCR、CD3D、LCP2、ITK、IL10以及p38基因。本研究为进一步了解异尖线虫和长江刀鲚之间的相互作用机制提供了数据支撑。进行了长江刀鲚血清代谢组分析。利用GC/MS技术对感染异尖线虫后的长江刀鲚血清进行非靶向代谢组学分析,共检测出65种差异代谢物,其中28种代谢物上调,37种代谢物下调。多元统计分析(PLS-DA和OPLS-DA)表明,异尖线虫感染对刀鲚血清代谢谱产生了显著(p<0.05)影响。对异尖线虫感染后的长江刀鲚血清中代谢物及其参与的代谢通路进行了分析,发现甘油酯代谢、生物素代谢、戊糖磷酸循环、丙氨酸代谢、d-谷氨酰胺和d-谷氨酸代谢、嘧啶代谢和三羧酸循环通路中有很多代谢物及调控相关代谢物合成的基因都出现了表达量变化。此外,还发现DAG的表达量显著上调,并在T细胞激活所需的关键免疫通路中发挥重要作用。
Abstract
chang jiang dao ji shi chang jiang xia you liu yu zui wei chong yao de bu lao he xiao fei dui xiang ,shi mu qian chang jiang liu yu wei yi neng xing cheng wen ding yu xun de jiang hai hui you xing yu lei 。zai shui yu sheng tai huan jing e hua ji ren lei bu lao xie pai de shuang chong ying xiang xia ,chang jiang dao ji zi yuan ri qu ku jie ,mu qian nian bu lao liang jin yao wei li shi zui gao ji lu de 2%。ben yan jiu ji tong diao cha chang jiang dao ji xian chong gan ran te zheng ji ji sheng xian chong qun la te zheng ;ji yu jian hua ji yin zu ji shu he er dan zhi wen yuan su ji shu shai shua chu wei chuan bei jing xiang shi 、hui you lv li xiang jin de chang jiang dao ji shi yan yang ben ,zeng ge li yong zhuai lu zu he dai xie zu ji shu yan jiu chang jiang dao ji gan ran yi jian xian chong hou xiang guan mian yi ji yin ji dai xie chan wu de bian hua ,jie shi yu gan ran yi jian xian chong xiang guan de mian yi xin hao tong lu ,tan tao hui you qun ti gan ran yi jian xian chong hou suo chan sheng de mian yi kuo ying xing fan ying 。jie ge chang jiang dao ji chong qun sheng tai xue yan jiu ,jin yi bu tan tao chang jiang dao ji yu ji sheng yi jian xian chong jian de xiang hu zuo yong ji zhi ,yan jiu jie guo dui yu hou xu yi jian xian chong gan ran dui chang jiang dao ji chong qun bu chong de ying xiang yan jiu ju you ji ji yi yi 。ben wen zhu yao yan jiu nei rong ru xia :ji tong diao cha le chang jiang dao ji gan ran xian chong qing kuang 。yu xun ji nei zai chang jiang kou zhi po yang hu de 7ge diao cha duan mian shang gong cai ji chang jiang dao ji 695wei ,cong 541wei yang ben zhong jian chu xian chong 7271tiao ,zong ti gan ran lv wei 77.84%,ping jun gan ran jiang du wei 13.44±30.68tiao /wei ,ping jun gan ran feng du wei 10.46±27.63tiao /wei ,bu tong duan mian jian dao ji yang ben xian chong gan ran jiang du he gan ran feng du cha yi xian zhe (p<0.05)。li yong fen zi jian ding fang fa dui sui ji chou yang de 1160tiao xian chong de jian ding jie guo xian shi ,gong jian chu yi jian ke xian chong 2shu 6chong ,fen bie wei pa shi yi jian xian chong (Anisakis pegreffii)、jian chan yi jian xian chong (A.simplex)、nei wan gong zhi xian chong (Hysterothylacium aduncum)、fei shi gong zhi xian chong (H.fabri)、zhong hua gong zhi xian chong (H.sinens)he sha men gong zhi xian chong (H.amoyense),ji zhong pa shi yi jian xian chong wei you shi chong ,zhan 84.84%。ITSxu lie fen xi jie guo xian shi ,pa shi yi jian xian chong de chan bei xing duo yang xing (Hd)wei 0.174,he gan suan duo yang xing (π)wei 0.00028,ge duan mian qun ti jian mei you fa sheng ming xian de wei chuan fen hua (Fst<0.05),zong ji yin liu (Nm)wei 75.74,qun ti jian ji yin jiao liu chong fen 。yan jiu le chang jiang dao ji chou yang qun ti wei chuan bei jing 。li yong 2b-RADji shu dui 165wei chang jiang dao ji yang ben jin hang ce xu ,jing shai shua he ju lei fen xi hou ,huo de 373244ge biao qian ,ji ping jun Uniquebiao qian shu wei 289602ge ,Uniquebiao qian bi dui lv bian fu wei 73.77%-80.25%。li yong SOAPruan jian jiang guo lv hou de Enzyme Readsbi dui dao can kao xu lie ,yi zui da shi ran fa (ML)jin hang SNPbiao ji fen xing ,gong huo de SNPbiao ji 36975ge ,ji zhong gao du duo tai xing de SNP(PIC>0.5)wei dian gong ji 26ge 。qun ti er tai xing SNPbiao ji 36469ge ,ji zhong yi A/Ghe C/Tzhuai huan de xing shi wei zhu ,fen bie you 10013ge he 12881ge ,fen bie zhan SNPzong liang de 27.08%he 34.84%;zhuai huan yu dian huan bi wei 1.69。fen zu jian ping jun guan ce za ge du Hobian hua fan wei wei 0.1286-0.1408,ping jun ji wang za ge du Hezui xiao wei 0.1340,zui da wei 0.1446,ge fen zu jian wei chuan fen hua ji shu jun di yu 0.005。biao ming chang jiang dao ji hui you qun ti wei chuan fen hua cheng du ji di ,ge ti xin xi jiao liu jin mi ,za ge du jiao gao ,ju you feng fu de wei chuan duo yang xing 。tong guo ben ci shai shua yan zheng ke bao zheng chang jiang dao ji zu xue shi yan yang ben ju you xiang shi de wei chuan bei jing ,jin er pai chu shi yan yang ben chong zhi cha yi dui fen xi jie guo de gan rao 。yan jiu le chang jiang dao ji chou yang qun ti sheng jing lv li 。li yong er dan zhi wen yuan su ji shu dui 81wei chang jiang dao ji yang ben de sheng jing lv li jin hang fan yan ,gong fa xian 3chong sheng tai lei xing :jiang -hai hui you xing (91.36%)、jiang -he kou hui you xing (6.17%)he dan shui ding ju xing (2.47%)。jiu gan ran xian chong yu sheng jing lv li de guan ji er yan ,suo you gan ran xian chong de chang jiang dao ji yang ben jun ju you jiang hai hui you lv li ,dan ju you jiang hai hui you lv li de chang jiang dao ji bu yi ding gan ran xian chong ,dan shui ding ju xing jun wei gan ran xian chong 。ben ci shai shua yan zheng ke bao zheng chang jiang dao ji zu xue yang ben ju you xiang jin de hui you lv li ,jin er pai chu shi yan yang ben yin qi xi sheng jing de cha yi dui fen xi jie guo chan sheng gan rao 。jin hang le chang jiang dao ji gan zang zhuai lu zu fen xi 。li yong RNA-seqji shu dui gan ran yi jian xian chong hou de chang jiang dao ji gan zang zu zhi jin hang biao da pu fen xi 。ce xu wan cheng hou ,dui yuan shi shu ju jin hang guo lv 、zu zhuang hou gong huo de 62604tiao Unigene。tong guo GOhe KEGGdeng sheng wu xin xi xue fang fa dui Unigenejin hang zhu shi he fen xi ,bing yu ce ji gong neng 。dui ji yin biao da cha yi fen xi ,gong huo de 961ge cha yi biao da ji yin ,bao gua 545ge shang diao ji yin he 416ge xia diao ji yin 。10ge su zhu ji yin qRT-PCRjian ce jie guo yu RNA-seqjie guo yi zhi ,jin yi bu zheng ming RNAce xu jie guo de zhen shi xing he ke kao xing 。tong guo pathwayfu ji fen xi ,shai shua chu mian yi xiang guan ji yin he mian yi xin hao tong lu ,bao gua can yu kang yuan jia gong he cheng di guo cheng de MHCⅡ、TCR、CTSLyi ji CIITAji yin he can yu Txi bao shou ti xin hao tong lu de TCR、CD3D、LCP2、ITK、IL10yi ji p38ji yin 。ben yan jiu wei jin yi bu le jie yi jian xian chong he chang jiang dao ji zhi jian de xiang hu zuo yong ji zhi di gong le shu ju zhi cheng 。jin hang le chang jiang dao ji xie qing dai xie zu fen xi 。li yong GC/MSji shu dui gan ran yi jian xian chong hou de chang jiang dao ji xie qing jin hang fei ba xiang dai xie zu xue fen xi ,gong jian ce chu 65chong cha yi dai xie wu ,ji zhong 28chong dai xie wu shang diao ,37chong dai xie wu xia diao 。duo yuan tong ji fen xi (PLS-DAhe OPLS-DA)biao ming ,yi jian xian chong gan ran dui dao ji xie qing dai xie pu chan sheng le xian zhe (p<0.05)ying xiang 。dui yi jian xian chong gan ran hou de chang jiang dao ji xie qing zhong dai xie wu ji ji can yu de dai xie tong lu jin hang le fen xi ,fa xian gan you zhi dai xie 、sheng wu su dai xie 、wu tang lin suan xun huan 、bing an suan dai xie 、d-gu an xian an he d-gu an suan dai xie 、mi ding dai xie he san suo suan xun huan tong lu zhong you hen duo dai xie wu ji diao kong xiang guan dai xie wu ge cheng de ji yin dou chu xian le biao da liang bian hua 。ci wai ,hai fa xian DAGde biao da liang xian zhe shang diao ,bing zai Txi bao ji huo suo xu de guan jian mian yi tong lu zhong fa hui chong yao zuo yong 。
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论文作者分别是来自安徽师范大学的刘凯，发表于刊物安徽师范大学2019-07-10论文，是一篇关于长江刀鲚论文,异尖线虫论文,耳石论文,元素指纹论文,组学研究论文,安徽师范大学2019-07-10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，资料来自安徽师范大学2019-07-10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。
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