本文主要研究内容
作者周渊涛（2019）在《亚洲小车蝗嗅觉相关蛋白的鉴定及功能研究》一文中研究指出：亚洲小车蝗Oedaleus asiaticus是中国北方草原和农牧交错带中最具优势和经济重要性的蝗虫种类之一,在爆发期间往往需要化学防治。该种蝗虫主要以禾本科植物为食,喜欢过度放牧的草原和旱生栖息地,是内蒙古草原退化的指示物种。昆虫主要以嗅觉感受系统识别生境中的化学气味分子,从而引导昆虫觅食、交配、选择产卵地点和躲避天地等生命活动。并且通过各种嗅觉蛋白之间的相互作用来检测气味分子,所以嗅觉相关蛋白是昆虫嗅觉活动的分子基础。然而,西前关于亚洲小车蝗嗅觉功能的研究信息很少,所以对其嗅觉相关蛋白基因的挖掘以及功能研究有利于明确其化学感受机制,同时也为找寻蝗虫绿色防治的分子靶标提供理论依据。本研究利用亚洲小车蝗触角转录组数据鉴定出嗅觉相关蛋白基因,对其进行生物信息学以及表达谱分析,并对主要的化学感受蛋白基因进行原核表达,然后测定该蛋白与不同配体的结合能力,最后通过RNA干扰结合触角电位对其干扰效应进行检测。上要结果如下:1.亚洲小车蝗嗅觉相关蛋白基因的鉴定根据本实验室组装的亚洲小车蝗触角转录组数据库,通过生物信息学方法分析,共鉴定出86个嗅觉相关蛋白白基因,包括17个化学感受蛋白基因(chemosensory protein,CSP)、60个气味受体基因(odorant receptor,OR)(包括59个普通气味受体基因OR和一个共受体ORco)、6个离子型受体基因(ionotropic receptor,IR)和3 个感觉神经元膜蛋白基因(sensory neuron membrane protein,SNMP)。17 个 CSPs中除OCsiCSP19外,其它16个OasiCSP基因具有完整的开放阅读框(open reading frame,ORF),其长度范围为372 bp至516 bp;氨基酸序列的多重比对表明这些OasiCSP含有4个保守的半胱氨酸残基。60个OR基因中只有OasiOR14、Oasi和R15和OasiORco具有完整的ORF,长度分别为1350 bp、1194 bp和1146 bp,它们编码的蛋白质含有4～7个跨膜结构域;6个IR具有1～4个跨膜结构域,除OasiIRI和OasiIR76b之外的其它4个OasilR具有16至19个氨基酸的信号肽,只有OasiIR8a和OasiIR25a具有全长的ORF;3个SNMPs有1～2个跨膜结构域,并且氨基酸序列比对发现SNMP含有6个保守的半胱氨酸残基。BlastP验证的最佳结果和系统发育分析均显示,这些嗅觉相关蛋白与来自直翅目的东亚飞蝗Locusta migaatoia或沙漠蝗Schier gregaria的嗅觉相关蛋白亲缘关系最近。2.亚洲小车蝗嗅觉相关蛋白基因的组织表达谱分析应用qRT-PCR分析嗅觉相关蛋白基因在雌雄成虫不同组织(触角、去除触角和口器的头、上唇、去掉下唇须的下唇、下唇须、下颚须、胸部、跗节、翅和腹部)中的表达模式。结果显示,8个CSP基因在成虫不同组织中的表达水平存在显著差异,特别是OasiCSP8在下唇须和下颚须中高表达,而OasiCSP11和OasiCSP13在触角中表达量最高。OasiCSp15在化学感受器官(触角、上唇、去掉下唇须的下唇、下唇须、下颚须和跗节)中的表达量远高于非化学感受器官(去掉触角和口器的头部、胸部、翅和腹部)中的表达量,而OasiCSp12在几乎所有测定的组织中具有相似的表达分布;14个OR和2个SNMP基因(OasiSNMP1和OasiSNMP2a)在成虫触角中强烈表达,并且几乎所有测试的基因在雌雄之间的表达水平具有显著差异。此外,2个IR基因(CasiIR25a和CasiIR76b)在触角,下唇须和下颚须中高表达,而3个IR基因(OasiIR1、OasiIR2和OasiIR3)在大多数组织中高表达(去掉触角和口器的头部、下唇须、下颚须、无下唇须的下唇、胸部、跗节和腹部),但OasiIR25a只在触角、去掉下唇须的下唇、下唇须、下颚须以及腹部中微弱表达。3.亚洲小车蝗化学感受蛋白荧光竞争结合测定构建了 8个OasiCSPs的重组表达质粒并在大肠杆菌中成功表达,用Ni亲和层析柱成功纯化蛋白,并测定了来自寄主植物克氏针茅Stipa krylovii挥发物、亚洲小车蝗粪便挥发物以及亚洲小车蝗成虫体表挥发物共16种气味化合物对这8种CSP蛋白的结合能力。结果表明,8种蛋白对各种挥发物表现出不同程度的结合能力,除了OasiCSP11对1种成虫体表挥发物(2-乙基己醇)表现出最高的结合能力(Ki值为9.84μM),OasiCSP12对大多数挥发物的结合能力高于其它7种蛋白,其中对5种寄主植物挥发物(正己醛、叶醇、乙酸丁酯、甲苯、三甲苯)和1种成虫体表挥发物(2-乙基己醇)表现出强的结合能力(Ki<20 μμM),尤其对乙酸丁酯和叶醇的结合力最强,Ki值分别为13.73与14.73 μM。OasiCSP15对供试化合物的结合力最弱,并且只结合2种气味化合物。4.亚洲小车蝗化学感受蛋白基因RNA干扰效应为进一步验证化学感受蛋白的嗅觉功能,进行了 RNA干扰(RNAinterference,RNAi)和电生理反应试验。在亚洲小车蝗雌雄成虫中分别注射3种dsCSPs(OasiCSP4-dsRNA、OasiCSP11-dsRNA 和 OasiCSP12-dsRNA)48 h后,qRT-PCR 检测显示,与注射水和非注射的对照组相比,注射dsCSP12后显著降低了雌雄触角中的靶基因表达水平,而在注射dsCSP4和dsCSP11后,OasiCSP4和OasiCSP111的表达水平在雌性中显著降低,但在雄性中没有显著差异。16种气味化合物对不同注射处理后的雌雄成虫进行触角电位检测显示,在dsCSP4处理后,雌触角只对3种化合物(反式-2-己烯醛、顺-3-己烯-1-醇和甲基丙烯醛)的触角电位(Electroantennogram,EAG)反应显著降低;雄触角对12种化合物的反应显著降低;并且在雌雄触角中,2-甲基丁醛在dsCSP4注射后反而引起更强的反应。沉默OasiCSP11后显著降低了雌触角对13种化合物的EAG反应,雄触角对4种化合物的反应显著降低。注射dsCSP12后雌雄触角对化合物的EAG反应最明显,雌触角对所有气味化合物的EAG反应显著降低,而雄触角除了对反式-2-己烯醛、顺-3-己烯-1-醇和庚醛外,对其它13种化合物的EAG反应也显著降低。上述结果表明,这3个CSP在亚洲小车蝗寻找寄主植物和聚集行为中可能起着重要作用。
Abstract
ya zhou xiao che huang Oedaleus asiaticusshi zhong guo bei fang cao yuan he nong mu jiao cuo dai zhong zui ju you shi he jing ji chong yao xing de huang chong chong lei zhi yi ,zai bao fa ji jian wang wang xu yao hua xue fang zhi 。gai chong huang chong zhu yao yi he ben ke zhi wu wei shi ,xi huan guo du fang mu de cao yuan he han sheng qi xi de ,shi nei meng gu cao yuan tui hua de zhi shi wu chong 。kun chong zhu yao yi xiu jiao gan shou ji tong shi bie sheng jing zhong de hua xue qi wei fen zi ,cong er yin dao kun chong mi shi 、jiao pei 、shua ze chan luan de dian he duo bi tian de deng sheng ming huo dong 。bing ju tong guo ge chong xiu jiao dan bai zhi jian de xiang hu zuo yong lai jian ce qi wei fen zi ,suo yi xiu jiao xiang guan dan bai shi kun chong xiu jiao huo dong de fen zi ji chu 。ran er ,xi qian guan yu ya zhou xiao che huang xiu jiao gong neng de yan jiu xin xi hen shao ,suo yi dui ji xiu jiao xiang guan dan bai ji yin de wa jue yi ji gong neng yan jiu you li yu ming que ji hua xue gan shou ji zhi ,tong shi ye wei zhao xun huang chong lu se fang zhi de fen zi ba biao di gong li lun yi ju 。ben yan jiu li yong ya zhou xiao che huang chu jiao zhuai lu zu shu ju jian ding chu xiu jiao xiang guan dan bai ji yin ,dui ji jin hang sheng wu xin xi xue yi ji biao da pu fen xi ,bing dui zhu yao de hua xue gan shou dan bai ji yin jin hang yuan he biao da ,ran hou ce ding gai dan bai yu bu tong pei ti de jie ge neng li ,zui hou tong guo RNAgan rao jie ge chu jiao dian wei dui ji gan rao xiao ying jin hang jian ce 。shang yao jie guo ru xia :1.ya zhou xiao che huang xiu jiao xiang guan dan bai ji yin de jian ding gen ju ben shi yan shi zu zhuang de ya zhou xiao che huang chu jiao zhuai lu zu shu ju ku ,tong guo sheng wu xin xi xue fang fa fen xi ,gong jian ding chu 86ge xiu jiao xiang guan dan bai bai ji yin ,bao gua 17ge hua xue gan shou dan bai ji yin (chemosensory protein,CSP)、60ge qi wei shou ti ji yin (odorant receptor,OR)(bao gua 59ge pu tong qi wei shou ti ji yin ORhe yi ge gong shou ti ORco)、6ge li zi xing shou ti ji yin (ionotropic receptor,IR)he 3 ge gan jiao shen jing yuan mo dan bai ji yin (sensory neuron membrane protein,SNMP)。17 ge CSPszhong chu OCsiCSP19wai ,ji ta 16ge OasiCSPji yin ju you wan zheng de kai fang yue dou kuang (open reading frame,ORF),ji chang du fan wei wei 372 bpzhi 516 bp;an ji suan xu lie de duo chong bi dui biao ming zhe xie OasiCSPhan you 4ge bao shou de ban guang an suan can ji 。60ge ORji yin zhong zhi you OasiOR14、Oasihe R15he OasiORcoju you wan zheng de ORF,chang du fen bie wei 1350 bp、1194 bphe 1146 bp,ta men bian ma de dan bai zhi han you 4～7ge kua mo jie gou yu ;6ge IRju you 1～4ge kua mo jie gou yu ,chu OasiIRIhe OasiIR76bzhi wai de ji ta 4ge OasilRju you 16zhi 19ge an ji suan de xin hao tai ,zhi you OasiIR8ahe OasiIR25aju you quan chang de ORF;3ge SNMPsyou 1～2ge kua mo jie gou yu ,bing ju an ji suan xu lie bi dui fa xian SNMPhan you 6ge bao shou de ban guang an suan can ji 。BlastPyan zheng de zui jia jie guo he ji tong fa yo fen xi jun xian shi ,zhe xie xiu jiao xiang guan dan bai yu lai zi zhi chi mu de dong ya fei huang Locusta migaatoiahuo sha mo huang Schier gregariade xiu jiao xiang guan dan bai qin yuan guan ji zui jin 。2.ya zhou xiao che huang xiu jiao xiang guan dan bai ji yin de zu zhi biao da pu fen xi ying yong qRT-PCRfen xi xiu jiao xiang guan dan bai ji yin zai ci xiong cheng chong bu tong zu zhi (chu jiao 、qu chu chu jiao he kou qi de tou 、shang chun 、qu diao xia chun xu de xia chun 、xia chun xu 、xia e xu 、xiong bu 、fu jie 、chi he fu bu )zhong de biao da mo shi 。jie guo xian shi ,8ge CSPji yin zai cheng chong bu tong zu zhi zhong de biao da shui ping cun zai xian zhe cha yi ,te bie shi OasiCSP8zai xia chun xu he xia e xu zhong gao biao da ,er OasiCSP11he OasiCSP13zai chu jiao zhong biao da liang zui gao 。OasiCSp15zai hua xue gan shou qi guan (chu jiao 、shang chun 、qu diao xia chun xu de xia chun 、xia chun xu 、xia e xu he fu jie )zhong de biao da liang yuan gao yu fei hua xue gan shou qi guan (qu diao chu jiao he kou qi de tou bu 、xiong bu 、chi he fu bu )zhong de biao da liang ,er OasiCSp12zai ji hu suo you ce ding de zu zhi zhong ju you xiang shi de biao da fen bu ;14ge ORhe 2ge SNMPji yin (OasiSNMP1he OasiSNMP2a)zai cheng chong chu jiao zhong jiang lie biao da ,bing ju ji hu suo you ce shi de ji yin zai ci xiong zhi jian de biao da shui ping ju you xian zhe cha yi 。ci wai ,2ge IRji yin (CasiIR25ahe CasiIR76b)zai chu jiao ,xia chun xu he xia e xu zhong gao biao da ,er 3ge IRji yin (OasiIR1、OasiIR2he OasiIR3)zai da duo shu zu zhi zhong gao biao da (qu diao chu jiao he kou qi de tou bu 、xia chun xu 、xia e xu 、mo xia chun xu de xia chun 、xiong bu 、fu jie he fu bu ),dan OasiIR25azhi zai chu jiao 、qu diao xia chun xu de xia chun 、xia chun xu 、xia e xu yi ji fu bu zhong wei ruo biao da 。3.ya zhou xiao che huang hua xue gan shou dan bai ying guang jing zheng jie ge ce ding gou jian le 8ge OasiCSPsde chong zu biao da zhi li bing zai da chang gan jun zhong cheng gong biao da ,yong Niqin he ceng xi zhu cheng gong chun hua dan bai ,bing ce ding le lai zi ji zhu zhi wu ke shi zhen mao Stipa kryloviihui fa wu 、ya zhou xiao che huang fen bian hui fa wu yi ji ya zhou xiao che huang cheng chong ti biao hui fa wu gong 16chong qi wei hua ge wu dui zhe 8chong CSPdan bai de jie ge neng li 。jie guo biao ming ,8chong dan bai dui ge chong hui fa wu biao xian chu bu tong cheng du de jie ge neng li ,chu le OasiCSP11dui 1chong cheng chong ti biao hui fa wu (2-yi ji ji chun )biao xian chu zui gao de jie ge neng li (Kizhi wei 9.84μM),OasiCSP12dui da duo shu hui fa wu de jie ge neng li gao yu ji ta 7chong dan bai ,ji zhong dui 5chong ji zhu zhi wu hui fa wu (zheng ji quan 、xie chun 、yi suan ding zhi 、jia ben 、san jia ben )he 1chong cheng chong ti biao hui fa wu (2-yi ji ji chun )biao xian chu jiang de jie ge neng li (Ki<20 μμM),you ji dui yi suan ding zhi he xie chun de jie ge li zui jiang ,Kizhi fen bie wei 13.73yu 14.73 μM。OasiCSP15dui gong shi hua ge wu de jie ge li zui ruo ,bing ju zhi jie ge 2chong qi wei hua ge wu 。4.ya zhou xiao che huang hua xue gan shou dan bai ji yin RNAgan rao xiao ying wei jin yi bu yan zheng hua xue gan shou dan bai de xiu jiao gong neng ,jin hang le RNAgan rao (RNAinterference,RNAi)he dian sheng li fan ying shi yan 。zai ya zhou xiao che huang ci xiong cheng chong zhong fen bie zhu she 3chong dsCSPs(OasiCSP4-dsRNA、OasiCSP11-dsRNA he OasiCSP12-dsRNA)48 hhou ,qRT-PCR jian ce xian shi ,yu zhu she shui he fei zhu she de dui zhao zu xiang bi ,zhu she dsCSP12hou xian zhe jiang di le ci xiong chu jiao zhong de ba ji yin biao da shui ping ,er zai zhu she dsCSP4he dsCSP11hou ,OasiCSP4he OasiCSP111de biao da shui ping zai ci xing zhong xian zhe jiang di ,dan zai xiong xing zhong mei you xian zhe cha yi 。16chong qi wei hua ge wu dui bu tong zhu she chu li hou de ci xiong cheng chong jin hang chu jiao dian wei jian ce xian shi ,zai dsCSP4chu li hou ,ci chu jiao zhi dui 3chong hua ge wu (fan shi -2-ji xi quan 、shun -3-ji xi -1-chun he jia ji bing xi quan )de chu jiao dian wei (Electroantennogram,EAG)fan ying xian zhe jiang di ;xiong chu jiao dui 12chong hua ge wu de fan ying xian zhe jiang di ;bing ju zai ci xiong chu jiao zhong ,2-jia ji ding quan zai dsCSP4zhu she hou fan er yin qi geng jiang de fan ying 。chen mo OasiCSP11hou xian zhe jiang di le ci chu jiao dui 13chong hua ge wu de EAGfan ying ,xiong chu jiao dui 4chong hua ge wu de fan ying xian zhe jiang di 。zhu she dsCSP12hou ci xiong chu jiao dui hua ge wu de EAGfan ying zui ming xian ,ci chu jiao dui suo you qi wei hua ge wu de EAGfan ying xian zhe jiang di ,er xiong chu jiao chu le dui fan shi -2-ji xi quan 、shun -3-ji xi -1-chun he geng quan wai ,dui ji ta 13chong hua ge wu de EAGfan ying ye xian zhe jiang di 。shang shu jie guo biao ming ,zhe 3ge CSPzai ya zhou xiao che huang xun zhao ji zhu zhi wu he ju ji hang wei zhong ke neng qi zhao chong yao zuo yong 。
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论文详细介绍
论文作者分别是来自内蒙古农业大学的周渊涛，发表于刊物内蒙古农业大学2019-10-08论文，是一篇关于亚洲小车蝗论文,嗅觉相关蛋白论文,表达谱论文,荧光竞争结合论文,干扰论文,反应论文,内蒙古农业大学2019-10-08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，资料来自内蒙古农业大学2019-10-08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：亚洲小车蝗论文; 嗅觉相关蛋白论文; 表达谱论文; 荧光竞争结合论文; 干扰论文; 反应论文; 内蒙古农业大学2019-10-08论文;