本文主要研究内容
作者孙红云（2019）在《苹果树腐烂病菌NRPS基因VmG10的功能研究》一文中研究指出：苹果树腐烂病作为一种枝干性真菌病害,极大地阻碍着我国苹果产业的健康发展,该病害发生面积广、为害程度重、防治困难,被称之为苹果树“癌症”。目前,苹果树腐烂病的研究主要集中在细胞壁降解酶、效应蛋白和次级代谢产物等方面。苹果树腐烂病菌(Valsa mali)全基因组测序结果显示,基因组里含有大量的NRPS基因,且有13个NRPS基因在病菌侵染枝条初期呈现明显的上调表达,这表明,NRPS基因在致病过程中扮演着重要角色。本研究前期通过敲除5个NRPS基因,随后通过致病力筛选,着重对NRPS基因Vm G10在生长发育和致病机理方面进行初步探究。主要研究内容及结果如下:1.运用q RT-PCR分析Vm G10在腐烂菌侵染枝条不同时间点的表达量,结果显示,同在野生型菌株03-8中表达量相比,Vm G10在腐烂菌侵染枝条6 h时上调表达7.12倍;随后通过PEG介导的遗传转化及引物检测得到敲除突变体2个、过表达突变体6个,通过一步克隆及最终引物检测得到回补突变体4个;2.观察PDA上菌丝生长速率及生长形态,发现VmG10的敲除影响菌丝生长形态,但不影响菌丝生长速率,敲除突变体ΔVm G10-19的菌丝边缘同03-8相比会产生更多的分支;产孢实验显示,同03-8相比,ΔVm G10-19产孢数下降约50%;致病力分析发现,ΔVm G10-19在叶片上致病力同03-8相比下降72.17%,在枝条上几乎不致病。过表达突变体致病力有不等程度的增强,其中Q15增加21.73%,Q16增加12.50%;黑色素表型观察及与黑色素转录调控相关6个基因定量结果显示,ΔVm G10-19中黑色素含量高于03-8,且ΔVm G10-19中与黑色素转录调控相关6个基因在菌丝生长2、3、4、5 d中的表达量均高于在03-8中的表达量;3.在0.5 M KCl,18 m M H2O2,200 mg/L CR,0.01%SDS四种非生物胁迫条件下,ΔVm G10-19比03-8受到的抑制程度更强,尤其是在18 m MH2O2条件下,抑制率较03-8提高44.44%;将03-8和ΔVm G10-19与苹果树上其他4种腐生菌做皿内对峙实验发现,ΔVm G10-19较03-8拮抗能力更弱,尤其在与苹果轮纹和炭疽对峙时;次级代谢产物分离结果显示,ΔVm G10-19发酵液处理过的烟草叶片产生的坏死斑较03-8发酵液处理过的烟草叶片产生的坏死斑面积减小32.8%,将发酵液稀释100倍再处理,结果基本一致;游离氨基酸含量测定结果显示,03-8发酵液中的天冬氨酸含量是ΔVm G10-19发酵液中的4倍,谷氨酸和精氨酸是其2倍;液相质谱鉴定发现,Vm G10的敲除的确影响了腐烂菌某种或某些次级代谢产物的合成,但不一定是短杆菌肽;发酵液处理莴苣种子,结果显示,小于1 KD的发酵液分离段处理过的莴苣种子,二者发芽率有明显差别,ΔVm G10-19发酵液小于1KD的分离段处理过的莴苣种子发芽率达63.3%,03-8发酵液小于1KD的分离段处理过的莴苣种子发芽率为23.7%,ddH2O处理的对照发芽率为86.7%。
Abstract
ping guo shu fu lan bing zuo wei yi chong zhi gan xing zhen jun bing hai ,ji da de zu ai zhao wo guo ping guo chan ye de jian kang fa zhan ,gai bing hai fa sheng mian ji an 、wei hai cheng du chong 、fang zhi kun nan ,bei chen zhi wei ping guo shu “ai zheng ”。mu qian ,ping guo shu fu lan bing de yan jiu zhu yao ji zhong zai xi bao bi jiang jie mei 、xiao ying dan bai he ci ji dai xie chan wu deng fang mian 。ping guo shu fu lan bing jun (Valsa mali)quan ji yin zu ce xu jie guo xian shi ,ji yin zu li han you da liang de NRPSji yin ,ju you 13ge NRPSji yin zai bing jun qin ran zhi tiao chu ji cheng xian ming xian de shang diao biao da ,zhe biao ming ,NRPSji yin zai zhi bing guo cheng zhong ban yan zhao chong yao jiao se 。ben yan jiu qian ji tong guo qiao chu 5ge NRPSji yin ,sui hou tong guo zhi bing li shai shua ,zhao chong dui NRPSji yin Vm G10zai sheng chang fa yo he zhi bing ji li fang mian jin hang chu bu tan jiu 。zhu yao yan jiu nei rong ji jie guo ru xia :1.yun yong q RT-PCRfen xi Vm G10zai fu lan jun qin ran zhi tiao bu tong shi jian dian de biao da liang ,jie guo xian shi ,tong zai ye sheng xing jun zhu 03-8zhong biao da liang xiang bi ,Vm G10zai fu lan jun qin ran zhi tiao 6 hshi shang diao biao da 7.12bei ;sui hou tong guo PEGjie dao de wei chuan zhuai hua ji yin wu jian ce de dao qiao chu tu bian ti 2ge 、guo biao da tu bian ti 6ge ,tong guo yi bu ke long ji zui zhong yin wu jian ce de dao hui bu tu bian ti 4ge ;2.guan cha PDAshang jun si sheng chang su lv ji sheng chang xing tai ,fa xian VmG10de qiao chu ying xiang jun si sheng chang xing tai ,dan bu ying xiang jun si sheng chang su lv ,qiao chu tu bian ti ΔVm G10-19de jun si bian yuan tong 03-8xiang bi hui chan sheng geng duo de fen zhi ;chan bao shi yan xian shi ,tong 03-8xiang bi ,ΔVm G10-19chan bao shu xia jiang yao 50%;zhi bing li fen xi fa xian ,ΔVm G10-19zai xie pian shang zhi bing li tong 03-8xiang bi xia jiang 72.17%,zai zhi tiao shang ji hu bu zhi bing 。guo biao da tu bian ti zhi bing li you bu deng cheng du de zeng jiang ,ji zhong Q15zeng jia 21.73%,Q16zeng jia 12.50%;hei se su biao xing guan cha ji yu hei se su zhuai lu diao kong xiang guan 6ge ji yin ding liang jie guo xian shi ,ΔVm G10-19zhong hei se su han liang gao yu 03-8,ju ΔVm G10-19zhong yu hei se su zhuai lu diao kong xiang guan 6ge ji yin zai jun si sheng chang 2、3、4、5 dzhong de biao da liang jun gao yu zai 03-8zhong de biao da liang ;3.zai 0.5 M KCl,18 m M H2O2,200 mg/L CR,0.01%SDSsi chong fei sheng wu xie pai tiao jian xia ,ΔVm G10-19bi 03-8shou dao de yi zhi cheng du geng jiang ,you ji shi zai 18 m MH2O2tiao jian xia ,yi zhi lv jiao 03-8di gao 44.44%;jiang 03-8he ΔVm G10-19yu ping guo shu shang ji ta 4chong fu sheng jun zuo min nei dui shi shi yan fa xian ,ΔVm G10-19jiao 03-8jie kang neng li geng ruo ,you ji zai yu ping guo lun wen he tan ju dui shi shi ;ci ji dai xie chan wu fen li jie guo xian shi ,ΔVm G10-19fa jiao ye chu li guo de yan cao xie pian chan sheng de huai si ban jiao 03-8fa jiao ye chu li guo de yan cao xie pian chan sheng de huai si ban mian ji jian xiao 32.8%,jiang fa jiao ye xi shi 100bei zai chu li ,jie guo ji ben yi zhi ;you li an ji suan han liang ce ding jie guo xian shi ,03-8fa jiao ye zhong de tian dong an suan han liang shi ΔVm G10-19fa jiao ye zhong de 4bei ,gu an suan he jing an suan shi ji 2bei ;ye xiang zhi pu jian ding fa xian ,Vm G10de qiao chu de que ying xiang le fu lan jun mou chong huo mou xie ci ji dai xie chan wu de ge cheng ,dan bu yi ding shi duan gan jun tai ;fa jiao ye chu li wo ju chong zi ,jie guo xian shi ,xiao yu 1 KDde fa jiao ye fen li duan chu li guo de wo ju chong zi ,er zhe fa ya lv you ming xian cha bie ,ΔVm G10-19fa jiao ye xiao yu 1KDde fen li duan chu li guo de wo ju chong zi fa ya lv da 63.3%,03-8fa jiao ye xiao yu 1KDde fen li duan chu li guo de wo ju chong zi fa ya lv wei 23.7%,ddH2Ochu li de dui zhao fa ya lv wei 86.7%。
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论文作者分别是来自西北农林科技大学的孙红云，发表于刊物西北农林科技大学2019-07-11论文，是一篇关于敲除论文,回补论文,毒力论文,生物活性检测论文,西北农林科技大学2019-07-11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，资料来自西北农林科技大学2019-07-11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：敲除论文; 回补论文; 毒力论文; 生物活性检测论文; 西北农林科技大学2019-07-11论文;