本文主要研究内容
作者刘培文（2019）在《白纹伊蚊雄性决定基因AalNix功能的研究与非缺陷型重组蚊浓核病毒的构建和效果测试》一文中研究指出：研究背景:伊蚊传播的疾病,如登革热,寨卡和基孔肯雅热,正在成为全球重大的公共卫生突发事件,而黄热病等旧的威胁也正在重新出现。白纹伊蚊作为一种重要的传病媒介,已经扩散到除南极洲以外的其他大陆,呈现全球扩散的严峻态势,已经成为最危险的100种入侵物种之一。由于缺乏安全有效的疫苗和特效药物,长期以来蚊媒种群防制是蚊媒传染病防控的关键部分,蚊媒种群防制主要有种群减少和种群替换。种群减少的主要手段就是通过施用化学杀虫剂来减少蚊媒种群或通过去除幼虫孳生地来防止蚊虫繁殖。然而随着化学杀虫剂的大量使用,蚊虫对杀虫剂抗性的相继升高,近年来蚊媒传染病的感染率逐年攀升,环境污染等问题也相继出现。由于蚊浓核病毒(Mosquitodensovirus,MDV)特异感染蚊类及其生活史的特性,让其具有成为高效安全生物杀蚊剂的潜力。发展MDV为新型生物杀蚊剂将为蚊媒传染病的生物防制提供一种新的思路。由于野生型MDV存在作用时间长,毒性弱的特点,对其进行基因重组,增强对蚊虫的致病性是一种可行的办法。前期的研究中,在MDV中引入了蝎毒基因和小干扰RNA以增强杀蚊效果,然而随着外源基因的引入,该病毒也丧失了自主复制能力,而这也是蚊浓核病毒发展为生物杀蚊剂的瓶颈。由于蚊媒传染病毒通过雌蚊吸血传播,控制雌蚊种群数量是蚊媒防制方法的核心,目前基于释放绝育转基因蚊减少野外蚊虫种群来控制登革热等蚊媒传染病的遗传策略已经进行了尝试并取得了成功。课题组前期研究已证实白纹伊蚊AalNix是雄性特异基因却具有复杂的基因结构,但是AalNix的功能还有待进一步研究。C6/36来自于雄性白纹伊蚊,然而C6/36细胞是否可作为性别决定机制研究的模型也尚不明确。目的:1.利用内含子内小RNA的表达策略,构建表达内源miRNA(Endengous miRNAs),miRNA 海绵(miRNA sponge)以及人工 miRNA(Artifitial miRNA,amiRNA)的MDV载体。重组MDV特异感染进入蚊细胞,分别诱导内源miRNA的过表达和沉默,以及诱导内源靶基因沉默。2.比较sgRNA在C6/36与白纹伊蚊胚胎中对AatNix的敲除效率;探究AalNix在C6/36细胞内是否参与调控性别决定通路,以明确C6/36细胞是否适合进行AalNix调控性别决定通路的研究;探究AalNix敲除是否导致雄性内生殖器的畸形和雌性化,进一步阐明AalNix在白纹伊蚊雄性发育中的功能。方法:1.利用内含子内小RNA的表达策略,分别构建表达miRNA,miRNA海绵和amiRNA的重组MDV载体。评估重组MDV中的人工内含子在蚊细胞内的剪接效率,并分别在蚊体内外验证重组MDV对内源性miRNA的过表达和沉默效果。透射电镜检测重组MDV病毒的完整性。PCR和DNA测序检测大肠杆菌stbl3株和C6/36细胞内重组MDV载体和重组MDV的遗传稳定性。qPCR检测重组MDV在C6/36细胞内以及白纹伊蚊幼虫孳生水体中的病毒增殖情况。2.针对AalNix外显子1设计sgRNA,构建同时表达相应sgRNA和Cas9的载体并转染入C6/36细胞测试各sgRNA的基因编辑效率。在AalNix中敲入RFP和Puromycin抗性基因,药筛和流式细胞术分选富集AalNix缺陷细胞,RT-qPCR检测aaldsxF和aalfruF的表达。体外合成的sgRNA与Cas9蛋白共注射白纹伊蚊胚胎,解剖性别表型异常成蚊观察其内生殖器形态,HRMA和测序验证AalNix的敲除效率,并分别检测doublesex和fruitless雌性和雄性剪接体的表达量。结果:1.成功构建了非缺陷型MDV载体和表达荧光的重组缺陷型MDV载体。重组病毒载体转染C6/36后,人工内含子可发生高效的剪切,感染蚊幼虫后,病毒可在体内播散。内含子内miRNA表达策略的MDV成功在蚊体内外分别促进了miR-210和let-7的高表达。内含子内miRNA海绵表达策略的MDV在蚊体内外分别诱导了 miR-210和let-7的沉默。内含子内amiRNA表达策略的MDV在蚊体内外诱导了靶基因V-ATpase基因沉默。重组MDV透射电镜下检测到完整病毒,感染C6/36和幼虫后,增殖能力与野生MDV无差异。重组MDV载体和重组MDV具有遗传稳定性。2.成功构建了 5个sgRNA的表达载体,其中sgRNA1和sgRNA2对AalNix具有基因编辑活性,sgRNAl的突变率为10.00%(4/40),sgRNA2的突变率为17.50%(7/40)。C6/36敲除AalNix后,aaldsxF和aalfruF的表达量均呈升高趋势。sgRNA1,sgRNA2和Cas9蛋白共注射白纹伊蚊胚胎,检测到36只畸形和雌性化表型的成蚊,其中大多数个体的的内生殖器也发生了畸形和雌性化,将其中20只进行HRMA,检测到1只sgRNA1位点熔解曲线异常,15只sgRNA2位点溶解曲线异常。测序的9只表型异常蚊中sgRNA2位点均存在突变,而sgRNA1位点突变仅有3只。sgRNA2的敲除效率高于sgRNA1,这与C6/36细胞中的效果一致。AalNix敲除后,aaldsx和aalfru均呈倾向雌性特异剪接的趋势。结论:1.重组MDV可以诱导内源性miRNA的过表达和沉默,以及通过表达amiRNA在蚊体内外诱导有效的基因沉默。重组MDV具有野生MDV般的增殖和二次传播能力。本研究首次构建了一种非缺陷型重组MDV的miRNA传递系统,该系统可作为蚊类基因功能分析的工具,为应用病毒的转基因共生体(viral paratransgenesis)进行蚊媒防制奠定了基础。2.针对AalNix的sgRNA1和sgRNA2在C6/36细胞和白纹伊蚊胚胎中具有一致的基因敲除效率。C6/36细胞可应用于AalNix对性别决定通路调控的研究。AalNix对雄性内生殖器的发育至关重要,进一步证实AalNix是雄性发育所必需的。尽管白纹伊蚊和埃及伊蚊的分化距离达到七千万年,但是Nix是一个保守的性别决定因子。
Abstract
yan jiu bei jing :yi wen chuan bo de ji bing ,ru deng ge re ,zhai ka he ji kong ken ya re ,zheng zai cheng wei quan qiu chong da de gong gong wei sheng tu fa shi jian ,er huang re bing deng jiu de wei xie ye zheng zai chong xin chu xian 。bai wen yi wen zuo wei yi chong chong yao de chuan bing mei jie ,yi jing kuo san dao chu na ji zhou yi wai de ji ta da liu ,cheng xian quan qiu kuo san de yan jun tai shi ,yi jing cheng wei zui wei xian de 100chong ru qin wu chong zhi yi 。you yu que fa an quan you xiao de yi miao he te xiao yao wu ,chang ji yi lai wen mei chong qun fang zhi shi wen mei chuan ran bing fang kong de guan jian bu fen ,wen mei chong qun fang zhi zhu yao you chong qun jian shao he chong qun ti huan 。chong qun jian shao de zhu yao shou duan jiu shi tong guo shi yong hua xue sha chong ji lai jian shao wen mei chong qun huo tong guo qu chu you chong zi sheng de lai fang zhi wen chong fan shi 。ran er sui zhao hua xue sha chong ji de da liang shi yong ,wen chong dui sha chong ji kang xing de xiang ji sheng gao ,jin nian lai wen mei chuan ran bing de gan ran lv zhu nian pan sheng ,huan jing wu ran deng wen ti ye xiang ji chu xian 。you yu wen nong he bing du (Mosquitodensovirus,MDV)te yi gan ran wen lei ji ji sheng huo shi de te xing ,rang ji ju you cheng wei gao xiao an quan sheng wu sha wen ji de qian li 。fa zhan MDVwei xin xing sheng wu sha wen ji jiang wei wen mei chuan ran bing de sheng wu fang zhi di gong yi chong xin de sai lu 。you yu ye sheng xing MDVcun zai zuo yong shi jian chang ,du xing ruo de te dian ,dui ji jin hang ji yin chong zu ,zeng jiang dui wen chong de zhi bing xing shi yi chong ke hang de ban fa 。qian ji de yan jiu zhong ,zai MDVzhong yin ru le xie du ji yin he xiao gan rao RNAyi zeng jiang sha wen xiao guo ,ran er sui zhao wai yuan ji yin de yin ru ,gai bing du ye sang shi le zi zhu fu zhi neng li ,er zhe ye shi wen nong he bing du fa zhan wei sheng wu sha wen ji de ping geng 。you yu wen mei chuan ran bing du tong guo ci wen xi xie chuan bo ,kong zhi ci wen chong qun shu liang shi wen mei fang zhi fang fa de he xin ,mu qian ji yu shi fang jue yo zhuai ji yin wen jian shao ye wai wen chong chong qun lai kong zhi deng ge re deng wen mei chuan ran bing de wei chuan ce lve yi jing jin hang le chang shi bing qu de le cheng gong 。ke ti zu qian ji yan jiu yi zheng shi bai wen yi wen AalNixshi xiong xing te yi ji yin que ju you fu za de ji yin jie gou ,dan shi AalNixde gong neng hai you dai jin yi bu yan jiu 。C6/36lai zi yu xiong xing bai wen yi wen ,ran er C6/36xi bao shi fou ke zuo wei xing bie jue ding ji zhi yan jiu de mo xing ye shang bu ming que 。mu de :1.li yong nei han zi nei xiao RNAde biao da ce lve ,gou jian biao da nei yuan miRNA(Endengous miRNAs),miRNA hai mian (miRNA sponge)yi ji ren gong miRNA(Artifitial miRNA,amiRNA)de MDVzai ti 。chong zu MDVte yi gan ran jin ru wen xi bao ,fen bie you dao nei yuan miRNAde guo biao da he chen mo ,yi ji you dao nei yuan ba ji yin chen mo 。2.bi jiao sgRNAzai C6/36yu bai wen yi wen pei tai zhong dui AatNixde qiao chu xiao lv ;tan jiu AalNixzai C6/36xi bao nei shi fou can yu diao kong xing bie jue ding tong lu ,yi ming que C6/36xi bao shi fou kuo ge jin hang AalNixdiao kong xing bie jue ding tong lu de yan jiu ;tan jiu AalNixqiao chu shi fou dao zhi xiong xing nei sheng shi qi de ji xing he ci xing hua ,jin yi bu chan ming AalNixzai bai wen yi wen xiong xing fa yo zhong de gong neng 。fang fa :1.li yong nei han zi nei xiao RNAde biao da ce lve ,fen bie gou jian biao da miRNA,miRNAhai mian he amiRNAde chong zu MDVzai ti 。ping gu chong zu MDVzhong de ren gong nei han zi zai wen xi bao nei de jian jie xiao lv ,bing fen bie zai wen ti nei wai yan zheng chong zu MDVdui nei yuan xing miRNAde guo biao da he chen mo xiao guo 。tou she dian jing jian ce chong zu MDVbing du de wan zheng xing 。PCRhe DNAce xu jian ce da chang gan jun stbl3zhu he C6/36xi bao nei chong zu MDVzai ti he chong zu MDVde wei chuan wen ding xing 。qPCRjian ce chong zu MDVzai C6/36xi bao nei yi ji bai wen yi wen you chong zi sheng shui ti zhong de bing du zeng shi qing kuang 。2.zhen dui AalNixwai xian zi 1she ji sgRNA,gou jian tong shi biao da xiang ying sgRNAhe Cas9de zai ti bing zhuai ran ru C6/36xi bao ce shi ge sgRNAde ji yin bian ji xiao lv 。zai AalNixzhong qiao ru RFPhe Puromycinkang xing ji yin ,yao shai he liu shi xi bao shu fen shua fu ji AalNixque xian xi bao ,RT-qPCRjian ce aaldsxFhe aalfruFde biao da 。ti wai ge cheng de sgRNAyu Cas9dan bai gong zhu she bai wen yi wen pei tai ,jie pou xing bie biao xing yi chang cheng wen guan cha ji nei sheng shi qi xing tai ,HRMAhe ce xu yan zheng AalNixde qiao chu xiao lv ,bing fen bie jian ce doublesexhe fruitlessci xing he xiong xing jian jie ti de biao da liang 。jie guo :1.cheng gong gou jian le fei que xian xing MDVzai ti he biao da ying guang de chong zu que xian xing MDVzai ti 。chong zu bing du zai ti zhuai ran C6/36hou ,ren gong nei han zi ke fa sheng gao xiao de jian qie ,gan ran wen you chong hou ,bing du ke zai ti nei bo san 。nei han zi nei miRNAbiao da ce lve de MDVcheng gong zai wen ti nei wai fen bie cu jin le miR-210he let-7de gao biao da 。nei han zi nei miRNAhai mian biao da ce lve de MDVzai wen ti nei wai fen bie you dao le miR-210he let-7de chen mo 。nei han zi nei amiRNAbiao da ce lve de MDVzai wen ti nei wai you dao le ba ji yin V-ATpaseji yin chen mo 。chong zu MDVtou she dian jing xia jian ce dao wan zheng bing du ,gan ran C6/36he you chong hou ,zeng shi neng li yu ye sheng MDVmo cha yi 。chong zu MDVzai ti he chong zu MDVju you wei chuan wen ding xing 。2.cheng gong gou jian le 5ge sgRNAde biao da zai ti ,ji zhong sgRNA1he sgRNA2dui AalNixju you ji yin bian ji huo xing ,sgRNAlde tu bian lv wei 10.00%(4/40),sgRNA2de tu bian lv wei 17.50%(7/40)。C6/36qiao chu AalNixhou ,aaldsxFhe aalfruFde biao da liang jun cheng sheng gao qu shi 。sgRNA1,sgRNA2he Cas9dan bai gong zhu she bai wen yi wen pei tai ,jian ce dao 36zhi ji xing he ci xing hua biao xing de cheng wen ,ji zhong da duo shu ge ti de de nei sheng shi qi ye fa sheng le ji xing he ci xing hua ,jiang ji zhong 20zhi jin hang HRMA,jian ce dao 1zhi sgRNA1wei dian rong jie qu xian yi chang ,15zhi sgRNA2wei dian rong jie qu xian yi chang 。ce xu de 9zhi biao xing yi chang wen zhong sgRNA2wei dian jun cun zai tu bian ,er sgRNA1wei dian tu bian jin you 3zhi 。sgRNA2de qiao chu xiao lv gao yu sgRNA1,zhe yu C6/36xi bao zhong de xiao guo yi zhi 。AalNixqiao chu hou ,aaldsxhe aalfrujun cheng qing xiang ci xing te yi jian jie de qu shi 。jie lun :1.chong zu MDVke yi you dao nei yuan xing miRNAde guo biao da he chen mo ,yi ji tong guo biao da amiRNAzai wen ti nei wai you dao you xiao de ji yin chen mo 。chong zu MDVju you ye sheng MDVban de zeng shi he er ci chuan bo neng li 。ben yan jiu shou ci gou jian le yi chong fei que xian xing chong zu MDVde miRNAchuan di ji tong ,gai ji tong ke zuo wei wen lei ji yin gong neng fen xi de gong ju ,wei ying yong bing du de zhuai ji yin gong sheng ti (viral paratransgenesis)jin hang wen mei fang zhi dian ding le ji chu 。2.zhen dui AalNixde sgRNA1he sgRNA2zai C6/36xi bao he bai wen yi wen pei tai zhong ju you yi zhi de ji yin qiao chu xiao lv 。C6/36xi bao ke ying yong yu AalNixdui xing bie jue ding tong lu diao kong de yan jiu 。AalNixdui xiong xing nei sheng shi qi de fa yo zhi guan chong yao ,jin yi bu zheng shi AalNixshi xiong xing fa yo suo bi xu de 。jin guan bai wen yi wen he ai ji yi wen de fen hua ju li da dao qi qian mo nian ,dan shi Nixshi yi ge bao shou de xing bie jue ding yin zi 。
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论文作者分别是来自南方医科大学的刘培文，发表于刊物南方医科大学2019-09-02论文，是一篇关于非缺陷型蚊浓核病毒论文,内含子论文,雄性决定因子论文,雌性化论文,南方医科大学2019-09-02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，资料来自南方医科大学2019-09-02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：非缺陷型蚊浓核病毒论文; 内含子论文; 雄性决定因子论文; 雌性化论文; 南方医科大学2019-09-02论文;