本文主要研究内容
作者张斌武（2019）在《苏云金芽胞杆菌Cry毒素蛋白抗蚜活性评价及其作用机理研究》一文中研究指出：苏云金芽胞杆菌(Bacillus thuringiensis,Bt)在其芽胞形成阶段产生的Cry毒素,是一类对多种农林害虫具有特异杀虫活性的毒素蛋白。cry基因已被广泛用于构建抗虫转基因作物,成为农业生产中防治鳞翅目、鞘翅目等Cry靶标昆虫的一种有效途径。随着这些转基因作物种植面积日益增大,对Cry不敏感的蚜虫占据了Cry靶标昆虫的原有生存空间,从次要害虫逐渐升格为主要害虫。目前控制蚜虫主要采用化学法,这与扩大转基因作物减少化学农药使用的目的背道而驰。因此,探究蚜虫对Cry毒素蛋白不敏感的原因,揭示Cry抗蚜机理具有重要的理论意义与应用前景。首先,本研究对不同Cry毒素的抗蚜活性进行评价。在大肠杆菌中表达了不同的Cry毒素:Cry1Ab1、Cry2Ab12、Cry7Ab4、Cry-related(一种Cry41同源蛋白,迄今抗蚜活性最高的Cry蛋白)和SCAN2-2(Cry46同源蛋白),并构建了cry2Ab12转基因烟草、cry-related转基因烟草、cry-related+Tma12双价转基因烟草(Tma12编码一种蕨类植物来源的几丁质酶)。利用膜囊法和叶圆片法分别考察Cry毒素及转基因烟草对桃蚜(Myzus persicae)的毒性水平,结果表明,Cry1Ab1蛋白对桃蚜基本没有毒性,SCAN2-2对桃蚜显示出较高的毒性,cry2Ab12转基因烟草对桃蚜低毒,cry-related转基因烟草及cry-related+Tma12双价转基因烟草均显示出较高抗蚜活性。此外,降落在双价转基因植株上的有翅蚜,全部死亡且没有发现其后代为害。其次,本研究采用Pull-down及LC-MS/MS技术,从桃蚜全虫匀浆上清中分离鉴定了Cry1Ab1、Cry7Ab4、Cry2Ab12、Cry-related、Cry1F及Vip3Aa的结合蛋白。结果表明,所有Cry毒素都存在与细胞吞噬相关的结合蛋白,这可能意味着Cry蛋白能够通过胞吞作用进入蚜虫细胞;所有Cry毒素都没有出现钙粘蛋白(CAD)及碱性磷酸酶(ALP)之类的结合蛋白(CAD和ALP是鳞翅目昆虫识别Cry的常见受体),这可能是Cry毒素对蚜虫低毒的原因;桃蚜Cathepsin-B及桃蚜共生菌来源的ATP-dependent-6-phosphofructokinase是Cry-related独有的结合蛋白,这可能是Cry-related抗蚜活性最高的原因。利用String数据库,对结合蛋白进行蛋白互作网络预测,结果显示部分Cry作用于蚜虫的分子过程涉及了MAPK、Hippo或Wnt通路。最后,本研究考察Cry-related与Cathepsin-B的体外互作,探讨Cathepsin-B在Cry-related抗蚜过程中的作用。在大肠杆菌中克隆表达了桃蚜Cathepsin-B蛋白,利用免疫印迹分析法证实Cry-related与Cathepsin-B二者体外可直接互作,这进一步表明Cry-related对蚜虫毒性最强可能是通过其与Cathepsin-B的互作来实现的。本研究还测定了Cathepsin-B的酶学性质,发现它是一个典型的半胱氨酸蛋白酶。半胱氨酸蛋白酶在细胞凋亡中起着重要作用,Cry-related属于能诱发细胞凋亡的抗癌晶体蛋白,所以,Cry-related与Cathepsin-B二者的互作,可能协同诱导细胞发生恶性凋亡。分子对接结果表明,Cry-related与Cathepsin-B的结合,封闭了Cathepsin-B的活性位点,这表明Cry-related还可能影响到蚜虫正常的蛋白降解,进而影响了蚜虫的生长发育。据此,本研究提出了Cry抗蚜机理假说:蚜虫的CAD、ALP与其他昆虫的存在显著差异,导致多数Cry毒素无法与其常见受体发生结合,因此对蚜虫不敏感,而Cry-related可以通过与Cathepsin-B的互作,诱发蚜虫细胞发生恶性凋亡或是影响蛋白降解利用进而发挥了抗蚜作用,Cry-related还可通过与共生菌来源的ATP-dependent-6-phosphofructokinase发生互作,影响蚜虫的生长发育,从而起到抗蚜作用。本研究结果,可为如何提高Cry抗蚜活性提供理论基础,对应用Cry有效防控蚜虫为害具有重要意义。
Abstract
su yun jin ya bao gan jun (Bacillus thuringiensis,Bt)zai ji ya bao xing cheng jie duan chan sheng de Crydu su ,shi yi lei dui duo chong nong lin hai chong ju you te yi sha chong huo xing de du su dan bai 。cryji yin yi bei an fan yong yu gou jian kang chong zhuai ji yin zuo wu ,cheng wei nong ye sheng chan zhong fang zhi lin chi mu 、qiao chi mu deng Cryba biao kun chong de yi chong you xiao tu jing 。sui zhao zhe xie zhuai ji yin zuo wu chong zhi mian ji ri yi zeng da ,dui Crybu min gan de ya chong zhan ju le Cryba biao kun chong de yuan you sheng cun kong jian ,cong ci yao hai chong zhu jian sheng ge wei zhu yao hai chong 。mu qian kong zhi ya chong zhu yao cai yong hua xue fa ,zhe yu kuo da zhuai ji yin zuo wu jian shao hua xue nong yao shi yong de mu de bei dao er chi 。yin ci ,tan jiu ya chong dui Crydu su dan bai bu min gan de yuan yin ,jie shi Crykang ya ji li ju you chong yao de li lun yi yi yu ying yong qian jing 。shou xian ,ben yan jiu dui bu tong Crydu su de kang ya huo xing jin hang ping jia 。zai da chang gan jun zhong biao da le bu tong de Crydu su :Cry1Ab1、Cry2Ab12、Cry7Ab4、Cry-related(yi chong Cry41tong yuan dan bai ,qi jin kang ya huo xing zui gao de Crydan bai )he SCAN2-2(Cry46tong yuan dan bai ),bing gou jian le cry2Ab12zhuai ji yin yan cao 、cry-relatedzhuai ji yin yan cao 、cry-related+Tma12shuang jia zhuai ji yin yan cao (Tma12bian ma yi chong jue lei zhi wu lai yuan de ji ding zhi mei )。li yong mo nang fa he xie yuan pian fa fen bie kao cha Crydu su ji zhuai ji yin yan cao dui tao ya (Myzus persicae)de du xing shui ping ,jie guo biao ming ,Cry1Ab1dan bai dui tao ya ji ben mei you du xing ,SCAN2-2dui tao ya xian shi chu jiao gao de du xing ,cry2Ab12zhuai ji yin yan cao dui tao ya di du ,cry-relatedzhuai ji yin yan cao ji cry-related+Tma12shuang jia zhuai ji yin yan cao jun xian shi chu jiao gao kang ya huo xing 。ci wai ,jiang la zai shuang jia zhuai ji yin zhi zhu shang de you chi ya ,quan bu si wang ju mei you fa xian ji hou dai wei hai 。ji ci ,ben yan jiu cai yong Pull-downji LC-MS/MSji shu ,cong tao ya quan chong yun jiang shang qing zhong fen li jian ding le Cry1Ab1、Cry7Ab4、Cry2Ab12、Cry-related、Cry1Fji Vip3Aade jie ge dan bai 。jie guo biao ming ,suo you Crydu su dou cun zai yu xi bao tun shi xiang guan de jie ge dan bai ,zhe ke neng yi wei zhao Crydan bai neng gou tong guo bao tun zuo yong jin ru ya chong xi bao ;suo you Crydu su dou mei you chu xian gai nian dan bai (CAD)ji jian xing lin suan mei (ALP)zhi lei de jie ge dan bai (CADhe ALPshi lin chi mu kun chong shi bie Cryde chang jian shou ti ),zhe ke neng shi Crydu su dui ya chong di du de yuan yin ;tao ya Cathepsin-Bji tao ya gong sheng jun lai yuan de ATP-dependent-6-phosphofructokinaseshi Cry-relateddu you de jie ge dan bai ,zhe ke neng shi Cry-relatedkang ya huo xing zui gao de yuan yin 。li yong Stringshu ju ku ,dui jie ge dan bai jin hang dan bai hu zuo wang lao yu ce ,jie guo xian shi bu fen Cryzuo yong yu ya chong de fen zi guo cheng she ji le MAPK、Hippohuo Wnttong lu 。zui hou ,ben yan jiu kao cha Cry-relatedyu Cathepsin-Bde ti wai hu zuo ,tan tao Cathepsin-Bzai Cry-relatedkang ya guo cheng zhong de zuo yong 。zai da chang gan jun zhong ke long biao da le tao ya Cathepsin-Bdan bai ,li yong mian yi yin ji fen xi fa zheng shi Cry-relatedyu Cathepsin-Ber zhe ti wai ke zhi jie hu zuo ,zhe jin yi bu biao ming Cry-relateddui ya chong du xing zui jiang ke neng shi tong guo ji yu Cathepsin-Bde hu zuo lai shi xian de 。ben yan jiu hai ce ding le Cathepsin-Bde mei xue xing zhi ,fa xian ta shi yi ge dian xing de ban guang an suan dan bai mei 。ban guang an suan dan bai mei zai xi bao diao wang zhong qi zhao chong yao zuo yong ,Cry-relatedshu yu neng you fa xi bao diao wang de kang ai jing ti dan bai ,suo yi ,Cry-relatedyu Cathepsin-Ber zhe de hu zuo ,ke neng xie tong you dao xi bao fa sheng e xing diao wang 。fen zi dui jie jie guo biao ming ,Cry-relatedyu Cathepsin-Bde jie ge ,feng bi le Cathepsin-Bde huo xing wei dian ,zhe biao ming Cry-relatedhai ke neng ying xiang dao ya chong zheng chang de dan bai jiang jie ,jin er ying xiang le ya chong de sheng chang fa yo 。ju ci ,ben yan jiu di chu le Crykang ya ji li jia shui :ya chong de CAD、ALPyu ji ta kun chong de cun zai xian zhe cha yi ,dao zhi duo shu Crydu su mo fa yu ji chang jian shou ti fa sheng jie ge ,yin ci dui ya chong bu min gan ,er Cry-relatedke yi tong guo yu Cathepsin-Bde hu zuo ,you fa ya chong xi bao fa sheng e xing diao wang huo shi ying xiang dan bai jiang jie li yong jin er fa hui le kang ya zuo yong ,Cry-relatedhai ke tong guo yu gong sheng jun lai yuan de ATP-dependent-6-phosphofructokinasefa sheng hu zuo ,ying xiang ya chong de sheng chang fa yo ,cong er qi dao kang ya zuo yong 。ben yan jiu jie guo ,ke wei ru he di gao Crykang ya huo xing di gong li lun ji chu ,dui ying yong Cryyou xiao fang kong ya chong wei hai ju you chong yao yi yi 。
论文参考文献
论文详细介绍
论文作者分别是来自华侨大学的张斌武，发表于刊物华侨大学2019-10-31论文，是一篇关于苏云金芽胞杆菌论文,蚜虫论文,结合蛋白论文,共生菌论文,作用机理论文,华侨大学2019-10-31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，资料来自华侨大学2019-10-31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：苏云金芽胞杆菌论文; 蚜虫论文; 结合蛋白论文; 共生菌论文; 作用机理论文; 华侨大学2019-10-31论文;