本文主要研究内容
作者李玲（2019）在《沙葱萤叶甲嗅觉相关蛋白的鉴定及功能研究》一文中研究指出：沙葱萤叶甲Galeruca daurica(Joannis)是一种近年来在内蒙古草原上暴发成灾的新害虫,危害沙葱Allium mongolium、野韭Allium ramosum、多根葱Allium polyrhizum等百合科Liliaceae葱属Alllim植物,严重破坏草原生态环境。嗅觉感受系统对昆虫的生命活动起到了至关重要的作用,昆虫通过嗅觉来感知外界环境中的挥发物质,从而进行信息交流。目前有关沙葱萤叶甲化学感受系统的研究还是空白。本研究从沙葱萤叶甲转录组中鉴定到大量的嗅觉相关基因,并对气味结合蛋白和化学感受蛋白基因的表达谱和原核表达嗅觉相关蛋白与寄主植物挥发物的结合特性进行了分析,为进一步研究沙葱萤叶甲化学感受的分子机理奠定了必要的基础,并对揭示其猖厥成灾机理以及开发利用引诱剂或驱避剂控制其发生危害具有重要的意义。主要研究结果如下:1.沙葱萤叶甲触角感器的显微观察利用扫描电子显微镜对沙葱萤叶甲触角上的感器进行了观察。结果表明,沙葱萤叶甲触角感器主要有5种类型,分别是毛形感器(sensillatrichodea)、刺形感器(sensilla chaetica)、锥形感器(sensilla basiconica)、钟形感器(sensilla campaniformia)和 Bohm 氏鬃毛(Bohm bristle)。2.沙葱萤叶甲嗅觉相关基因的鉴定及分子特性分析从本实验室组装的沙葱萤叶甲转录组中鉴定到66条嗅觉相关基因,包括29条气味结合蛋白(odorant binding protein,OBP)基因、10条化学感受蛋白(chemosensory protein,CSP)基因、21条嗅觉受体(olfactory receptor,OR)基因(包括1条嗅觉共同受体 olfactory receptor co-receptor,ORco)和6条感觉神经元膜蛋白(sensory neuron membrane protein,SNMP)基因。序列分析结果表明,除OBP29外,其它28个OBP基因均具有完整的开放阅读编码框(open reading frame,ORF),编码氨基酸个数119～202。根据保守的半胱氨酸残基数可将GdauOBP分为Classic OBP和Minus-C OBP两个亚家族。10条GdauCSP基因均具有完整的ORF,编码氨基酸个数98～273,序列中含有四个保守的半胱氨酸。GdauOR基因的ORF均不完整,编码55～259个氨基酸。6个GdauSNMP中,GdauSNMPla和GdauSNMP1b具有完整的ORF,均含有2个跨膜结构域,序列中含有6个保守的半胱氨酸残基。其它4个GdauSNMP基因的ORF不完整,序列中只含有1个跨膜结构域。BlastX验证的最佳结果和系统发育分析均显示,沙葱萤叶甲与大猿叶甲Colaphellus bowringi、榆黄毛萤叶甲Pyrrhalta maculicollis、榆绿毛萤叶甲Pyrrhaltaaenescens等鞘翅目昆虫嗅觉蛋白的亲缘关系最近。GdauORco在系统进化树中与鳞翅目、双翅目、半翅目、直翅目以及鞘翅目其它昆虫的ORco独立地形成一支,表现出极度保守的进化机制。3.沙葱萤叶甲OBP和CSP基因的时空表达格局通过半定量和实时荧光定量技术对沙葱萤叶甲OBP和CSP基因的表达谱进行了分析。结果表明,GdauOBP和GdauCSP基因不仅表达于成虫触角中,还广泛表达于足、翅、头、胸、腹等各部位。各基因在卵、幼虫、蛹和成虫等各发育阶段也有不同程度的表达。超过一半的基因在成虫触角中的表达量最高,且雌雄触角之间的表达水平存在显著差异。同时,大多数GdauOBP和GdauCSP基因在卵和蛹中的表达水平相对较低。4.沙葱萤叶甲OBP和CSP的克隆、原核表达及纯化通过 RACE 技术克隆得到 GdauOBP1、GdauOBBP6、GdauOBP10、GdauOBP20、GdauCSP4和GdauCSP5的cDNA全长序列以及GdauOBP15的ORF序列和3’非编码区,验证了 ORF的完整性。成功构建了重组表达质粒pET-28a(+)/OBP1、pET-28a(+)/OBP6、pET-28a(+)/OBP10、pET-28a(+)/OBP15、pET-28a(+)/OBP20、pET-28a(+)/CSP4和pET-28a(+)/CSP5,通过诱导表达和蛋白纯化得到了可溶性重组蛋白。5.沙葱萤叶甲主要寄主植物挥发物的鉴定及其触角电位反应采用顶空动态收集法和GC-MS技术从沙葱萤叶甲最适寄主植物—沙葱中鉴定出32种挥发性化合物,并从中选取了 13种主要化合物进行EAG测定。EAG测试结果表明,二烯丙基硫醚、二烯丙基二硫、二烯丙基三硫醚、顺-2-己烯-1-醇、2-己烯醛、苯甲酸甲酯和己醛7种化合物能引起沙葱萤叶甲触角较为强烈的电位反应。此外,雌性对多数化合物的反应值均高于雄性;当化合物浓度在0.1 mol/L和1 mol/L时,沙葱萤叶甲的触角电位反应较高。6.沙葱萤叶甲OBP和CSP与主要寄主植物挥发物的结合特性荧光竞争结合试验以N-苯基-1-萘胺(N-phenyl-1-naphthylamine,1-NPN)为探针。结果表明,GdauOBP1和GdauOBP1O对所有供试的13种寄主植物挥发物结合能力均较弱(Ki>30μM);GdauOBP6与二甲基二硫醚、己醛、2-己烯醛和顺-2-己烯-1-醇有较强的结合能力,Ki值为 25.65～29.09μM;GdauOBP15特异性地结合二甲基二硫醚,K值为23.09μM;GdauOBP20仅与对二甲苯和环庚三烯有较强的结合能力,Ki值分别为22.91 μM和26.55 μM。两个CSP对寄主植物挥发物的结合能力差异很大,GdauCSP4对所测化合物具有广谱的结合能力,能与其中9种配体较好地结合,K值为12.06 μM～21.32 μM,其中结合能力最强的配体是苯甲酸甲酯,其次为己醛;而GdauCSP5的结合谱较窄,仅能较好地结合二甲基二硫醚和2-己烯醛,Ki值分别为26.47μM和25.28 μM。所有7个重组蛋白与二烯丙基硫醚、月桂烯和二烯丙基三硫醚的结合能力均较弱(Ki>30 μM)。
Abstract
sha cong ying xie jia Galeruca daurica(Joannis)shi yi chong jin nian lai zai nei meng gu cao yuan shang bao fa cheng zai de xin hai chong ,wei hai sha cong Allium mongolium、ye jiu Allium ramosum、duo gen cong Allium polyrhizumdeng bai ge ke Liliaceaecong shu Alllimzhi wu ,yan chong po huai cao yuan sheng tai huan jing 。xiu jiao gan shou ji tong dui kun chong de sheng ming huo dong qi dao le zhi guan chong yao de zuo yong ,kun chong tong guo xiu jiao lai gan zhi wai jie huan jing zhong de hui fa wu zhi ,cong er jin hang xin xi jiao liu 。mu qian you guan sha cong ying xie jia hua xue gan shou ji tong de yan jiu hai shi kong bai 。ben yan jiu cong sha cong ying xie jia zhuai lu zu zhong jian ding dao da liang de xiu jiao xiang guan ji yin ,bing dui qi wei jie ge dan bai he hua xue gan shou dan bai ji yin de biao da pu he yuan he biao da xiu jiao xiang guan dan bai yu ji zhu zhi wu hui fa wu de jie ge te xing jin hang le fen xi ,wei jin yi bu yan jiu sha cong ying xie jia hua xue gan shou de fen zi ji li dian ding le bi yao de ji chu ,bing dui jie shi ji chang jue cheng zai ji li yi ji kai fa li yong yin you ji huo qu bi ji kong zhi ji fa sheng wei hai ju you chong yao de yi yi 。zhu yao yan jiu jie guo ru xia :1.sha cong ying xie jia chu jiao gan qi de xian wei guan cha li yong sao miao dian zi xian wei jing dui sha cong ying xie jia chu jiao shang de gan qi jin hang le guan cha 。jie guo biao ming ,sha cong ying xie jia chu jiao gan qi zhu yao you 5chong lei xing ,fen bie shi mao xing gan qi (sensillatrichodea)、ci xing gan qi (sensilla chaetica)、zhui xing gan qi (sensilla basiconica)、zhong xing gan qi (sensilla campaniformia)he Bohm shi zong mao (Bohm bristle)。2.sha cong ying xie jia xiu jiao xiang guan ji yin de jian ding ji fen zi te xing fen xi cong ben shi yan shi zu zhuang de sha cong ying xie jia zhuai lu zu zhong jian ding dao 66tiao xiu jiao xiang guan ji yin ,bao gua 29tiao qi wei jie ge dan bai (odorant binding protein,OBP)ji yin 、10tiao hua xue gan shou dan bai (chemosensory protein,CSP)ji yin 、21tiao xiu jiao shou ti (olfactory receptor,OR)ji yin (bao gua 1tiao xiu jiao gong tong shou ti olfactory receptor co-receptor,ORco)he 6tiao gan jiao shen jing yuan mo dan bai (sensory neuron membrane protein,SNMP)ji yin 。xu lie fen xi jie guo biao ming ,chu OBP29wai ,ji ta 28ge OBPji yin jun ju you wan zheng de kai fang yue dou bian ma kuang (open reading frame,ORF),bian ma an ji suan ge shu 119～202。gen ju bao shou de ban guang an suan can ji shu ke jiang GdauOBPfen wei Classic OBPhe Minus-C OBPliang ge ya jia zu 。10tiao GdauCSPji yin jun ju you wan zheng de ORF,bian ma an ji suan ge shu 98～273,xu lie zhong han you si ge bao shou de ban guang an suan 。GdauORji yin de ORFjun bu wan zheng ,bian ma 55～259ge an ji suan 。6ge GdauSNMPzhong ,GdauSNMPlahe GdauSNMP1bju you wan zheng de ORF,jun han you 2ge kua mo jie gou yu ,xu lie zhong han you 6ge bao shou de ban guang an suan can ji 。ji ta 4ge GdauSNMPji yin de ORFbu wan zheng ,xu lie zhong zhi han you 1ge kua mo jie gou yu 。BlastXyan zheng de zui jia jie guo he ji tong fa yo fen xi jun xian shi ,sha cong ying xie jia yu da yuan xie jia Colaphellus bowringi、yu huang mao ying xie jia Pyrrhalta maculicollis、yu lu mao ying xie jia Pyrrhaltaaenescensdeng qiao chi mu kun chong xiu jiao dan bai de qin yuan guan ji zui jin 。GdauORcozai ji tong jin hua shu zhong yu lin chi mu 、shuang chi mu 、ban chi mu 、zhi chi mu yi ji qiao chi mu ji ta kun chong de ORcodu li de xing cheng yi zhi ,biao xian chu ji du bao shou de jin hua ji zhi 。3.sha cong ying xie jia OBPhe CSPji yin de shi kong biao da ge ju tong guo ban ding liang he shi shi ying guang ding liang ji shu dui sha cong ying xie jia OBPhe CSPji yin de biao da pu jin hang le fen xi 。jie guo biao ming ,GdauOBPhe GdauCSPji yin bu jin biao da yu cheng chong chu jiao zhong ,hai an fan biao da yu zu 、chi 、tou 、xiong 、fu deng ge bu wei 。ge ji yin zai luan 、you chong 、yong he cheng chong deng ge fa yo jie duan ye you bu tong cheng du de biao da 。chao guo yi ban de ji yin zai cheng chong chu jiao zhong de biao da liang zui gao ,ju ci xiong chu jiao zhi jian de biao da shui ping cun zai xian zhe cha yi 。tong shi ,da duo shu GdauOBPhe GdauCSPji yin zai luan he yong zhong de biao da shui ping xiang dui jiao di 。4.sha cong ying xie jia OBPhe CSPde ke long 、yuan he biao da ji chun hua tong guo RACE ji shu ke long de dao GdauOBP1、GdauOBBP6、GdauOBP10、GdauOBP20、GdauCSP4he GdauCSP5de cDNAquan chang xu lie yi ji GdauOBP15de ORFxu lie he 3’fei bian ma ou ,yan zheng le ORFde wan zheng xing 。cheng gong gou jian le chong zu biao da zhi li pET-28a(+)/OBP1、pET-28a(+)/OBP6、pET-28a(+)/OBP10、pET-28a(+)/OBP15、pET-28a(+)/OBP20、pET-28a(+)/CSP4he pET-28a(+)/CSP5,tong guo you dao biao da he dan bai chun hua de dao le ke rong xing chong zu dan bai 。5.sha cong ying xie jia zhu yao ji zhu zhi wu hui fa wu de jian ding ji ji chu jiao dian wei fan ying cai yong ding kong dong tai shou ji fa he GC-MSji shu cong sha cong ying xie jia zui kuo ji zhu zhi wu —sha cong zhong jian ding chu 32chong hui fa xing hua ge wu ,bing cong zhong shua qu le 13chong zhu yao hua ge wu jin hang EAGce ding 。EAGce shi jie guo biao ming ,er xi bing ji liu mi 、er xi bing ji er liu 、er xi bing ji san liu mi 、shun -2-ji xi -1-chun 、2-ji xi quan 、ben jia suan jia zhi he ji quan 7chong hua ge wu neng yin qi sha cong ying xie jia chu jiao jiao wei jiang lie de dian wei fan ying 。ci wai ,ci xing dui duo shu hua ge wu de fan ying zhi jun gao yu xiong xing ;dang hua ge wu nong du zai 0.1 mol/Lhe 1 mol/Lshi ,sha cong ying xie jia de chu jiao dian wei fan ying jiao gao 。6.sha cong ying xie jia OBPhe CSPyu zhu yao ji zhu zhi wu hui fa wu de jie ge te xing ying guang jing zheng jie ge shi yan yi N-ben ji -1-nai an (N-phenyl-1-naphthylamine,1-NPN)wei tan zhen 。jie guo biao ming ,GdauOBP1he GdauOBP1Odui suo you gong shi de 13chong ji zhu zhi wu hui fa wu jie ge neng li jun jiao ruo (Ki>30μM);GdauOBP6yu er jia ji er liu mi 、ji quan 、2-ji xi quan he shun -2-ji xi -1-chun you jiao jiang de jie ge neng li ,Kizhi wei 25.65～29.09μM;GdauOBP15te yi xing de jie ge er jia ji er liu mi ,Kzhi wei 23.09μM;GdauOBP20jin yu dui er jia ben he huan geng san xi you jiao jiang de jie ge neng li ,Kizhi fen bie wei 22.91 μMhe 26.55 μM。liang ge CSPdui ji zhu zhi wu hui fa wu de jie ge neng li cha yi hen da ,GdauCSP4dui suo ce hua ge wu ju you an pu de jie ge neng li ,neng yu ji zhong 9chong pei ti jiao hao de jie ge ,Kzhi wei 12.06 μM～21.32 μM,ji zhong jie ge neng li zui jiang de pei ti shi ben jia suan jia zhi ,ji ci wei ji quan ;er GdauCSP5de jie ge pu jiao zhai ,jin neng jiao hao de jie ge er jia ji er liu mi he 2-ji xi quan ,Kizhi fen bie wei 26.47μMhe 25.28 μM。suo you 7ge chong zu dan bai yu er xi bing ji liu mi 、yue gui xi he er xi bing ji san liu mi de jie ge neng li jun jiao ruo (Ki>30 μM)。
论文参考文献
论文详细介绍
论文作者分别是来自内蒙古农业大学的李玲，发表于刊物内蒙古农业大学2019-10-08论文，是一篇关于沙葱萤叶甲论文,嗅觉相关蛋白论文,表达谱论文,分子克隆论文,原核表达论文,荧光竞争结合试验论文,触角电位论文,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：沙葱萤叶甲论文; 嗅觉相关蛋白论文; 表达谱论文; 分子克隆论文; 原核表达论文; 荧光竞争结合试验论文; 触角电位论文; 内蒙古农业大学2019-10-08论文;