本文主要研究内容
作者孙翠（2019）在《酵母细胞壁对梨和番茄果实采后病原真菌抗性的诱导作用及相关机理研究》一文中研究指出：真菌病害是引起果蔬采后巨大损失的原因之一。利用拮抗酵母抑制果实采后病害,被认为是一种最有希望替代化学杀菌剂的新型病害防治技术,受到了国内外越来越多的关注和研究。通过近30年的研究表明,生防酵母的拮抗机理主要包括营养与空间竞争、诱导果实抗性、分泌水解酶和吸附作用等。在这些机理中,诱导宿主抗性被认为是抑制病原菌的重要因素之一,其与果实的多种生理代谢活动密切相关。本课题组在前期研究中初步探明了拮抗酵母灭活菌体及其细胞壁能够诱导梨果实对青霉病的抗性,但拮抗酵母细胞壁诱导果实抗性的分子机理尚不明确。本论文主要以酵母细胞壁与梨果实和番茄果实为研究对象,探讨了酵母细胞壁诱导果实抗性依赖的物质基础及其相关的生物学机制。主要研究结果如下:(1)酵母细胞壁诱导梨果实青霉病抗性的物质基础分析对比分析了8株拮抗酵母、酿酒酵母、毕赤酵母的活体细胞、灭活菌体和酵母细胞壁对果实病害的抑制效力,研究表明:除了酿酒酵母和毕赤酵母外,8株拮抗酵母均能够大幅度的降低梨果实采后青霉病的发生;同时,灭活的酵母菌体及酵母细胞壁均能诱导梨果实对青霉病的抗性,其中以海洋红冬孢酵母(Rhodosporidium paludigenum)的灭活菌体和细胞壁的效果最佳。对酵母细胞壁组分分析表明,不同来源的同种酵母细胞壁组成上具有高度相似性,但不同种拮抗酵母的细胞壁组成上差异较大。进一步对细胞壁多糖组分的分析表明,来源于酿酒酵母和红冬孢酵母细胞壁的几丁质、β-1,3-D-葡聚糖和β-1,6-D-葡聚糖均能诱导梨果实产生对青霉病的抗性,而甘露糖蛋白对梨果实青霉病的抗性没有显著的影响。(2)酿酒酵母细胞壁几丁质的结构鉴定及对番茄果实抗性的诱导作用采用乌氏粘度计、高效液相色谱(HPLC)、傅里叶红外光谱(FTIR)和核磁共振(NMR)对酿酒酵母细胞壁几丁质的结构进行了表征,并探究了其对番茄灰霉病的防治效力及相关机理。研究结果表明,酵母细胞壁几丁质由单糖N-乙酰氨基葡萄糖组成,分子量为4.68×10~5 Dalton,脱乙酰度为45.83%。几丁质不能直接抑制灰葡萄孢(Botrytis cinerea)的生长,但可以显著诱导番茄果实对由B.cinerea引起的灰霉病的抵抗能力,且诱导效果与处理浓度和诱导时间密切相关。进一步的分析结果表明,酵母细胞壁几丁质诱导了果实组织中活性氧(ROS)的积累和胼胝质的沉积;同时提高了果实防御相关酶(SOD,CAT,POD,PAL,GLU和CHI)活性的增加及其相应的基因的上调;而且酵母细胞壁几丁质处理还激活了水杨酸信号通路关键基因(ICS,NPR1,TGA1a,TGA2,PR1b1和PR5)的上调表达。(3)酿酒酵母细胞壁β-1,3-D-葡聚糖的结构鉴定及生防效力评价通过乌氏粘度计、HPLC、FTIR和NMR等技术手段对β-1,3-D-葡聚糖的分子量(M_w)、单糖组成、特征性官能团和碳氢信号进行了表征。结果表明,分离得到的β-1,3-D-葡聚糖样品由单糖葡萄糖组成,分子量为165 kDa,葡萄糖分子间通过β-1,3糖苷键连接。在诱导果实抗性方面,β-1,3-D-葡聚糖不能直接影响扩展青霉(Penicillium expansum)孢子在体外和果实伤口处的萌发,而β-1,3-D-葡聚糖诱导处理显著抑制了果实伤口处P.expansum孢子的萌发。扫描电镜(SEM)观察β-1,3-D-葡聚糖处理对梨果实伤口组织超微结构影响的结果表明,β-1,3-D-葡聚糖可以在细胞组织水平上提高对P.expansum的抗性。RT-qPCR结果表明,β-1,3-D-葡聚糖处理显著提高了果实伤口周围组织中PR1、GLU、endoGLU9、CHI3、CHI4、endoCHI、PR4、PR5和PAL基因的上调表达,进而降低了梨果实青霉病腐烂的发病率和病斑直径。(4)酵母细胞壁葡聚糖合成相关基因Rho1的遗传转化及其生防效力评价利用特异性引物从酿酒酵母基因组DNA中扩增得到目的基因Rho1全长序列,克隆至载体pYES6/CT后,通过电转化技术导入至酿酒酵母中,测序鉴定后成功得到过表达酿酒酵母菌株SC/Rho1。RT-qPCR结果显示,诱导培养12 h后重组子SC/Rho1中Rho1基因的表达量较野生型菌株上调了107.89倍。同时,重组子SC/Rho1细胞壁中葡聚糖含量增加了9.26%。诱导抗性结果表明,诱导培养后的酵母细胞壁显示出对梨果实更强的诱导效果。(5)酵母细胞壁β-1,3-D-葡聚糖对诱导梨果实青霉病抗性的转录组学分析对β-1,3-D-葡聚糖和病原菌处理后的梨果实组织进行转录组学分析。结果表明,经酿酒酵母细胞壁β-1,3-D-葡聚糖与病原菌处理后的梨果肉组织相比于对照组有5984个基因上调表达,8769个基因下调表达。β-1,3-D-葡聚糖可能通过激活了梨果实细胞中模式分子识别受体FLS2、CNGCs和钙离子信号通道,引发了下游的激素信号生长素、茉莉酸、赤霉素和乙烯的传导,导致防御相关转录因子(MYC2、PTI5、PTI6、WRKY29和WRKY33)的表达,进而产生一系列的防御反应,包括防御相关基因POD、PAL、HSP90和PR1的变化、氨基酸的生物合成和代谢,以及次级代谢通路的激活,从而抑制了梨果实青霉病的发生和发展。
Abstract
zhen jun bing hai shi yin qi guo shu cai hou ju da sun shi de yuan yin zhi yi 。li yong jie kang jiao mu yi zhi guo shi cai hou bing hai ,bei ren wei shi yi chong zui you xi wang ti dai hua xue sha jun ji de xin xing bing hai fang zhi ji shu ,shou dao le guo nei wai yue lai yue duo de guan zhu he yan jiu 。tong guo jin 30nian de yan jiu biao ming ,sheng fang jiao mu de jie kang ji li zhu yao bao gua ying yang yu kong jian jing zheng 、you dao guo shi kang xing 、fen bi shui jie mei he xi fu zuo yong deng 。zai zhe xie ji li zhong ,you dao su zhu kang xing bei ren wei shi yi zhi bing yuan jun de chong yao yin su zhi yi ,ji yu guo shi de duo chong sheng li dai xie huo dong mi qie xiang guan 。ben ke ti zu zai qian ji yan jiu zhong chu bu tan ming le jie kang jiao mu mie huo jun ti ji ji xi bao bi neng gou you dao li guo shi dui qing mei bing de kang xing ,dan jie kang jiao mu xi bao bi you dao guo shi kang xing de fen zi ji li shang bu ming que 。ben lun wen zhu yao yi jiao mu xi bao bi yu li guo shi he fan jia guo shi wei yan jiu dui xiang ,tan tao le jiao mu xi bao bi you dao guo shi kang xing yi lai de wu zhi ji chu ji ji xiang guan de sheng wu xue ji zhi 。zhu yao yan jiu jie guo ru xia :(1)jiao mu xi bao bi you dao li guo shi qing mei bing kang xing de wu zhi ji chu fen xi dui bi fen xi le 8zhu jie kang jiao mu 、niang jiu jiao mu 、bi chi jiao mu de huo ti xi bao 、mie huo jun ti he jiao mu xi bao bi dui guo shi bing hai de yi zhi xiao li ,yan jiu biao ming :chu le niang jiu jiao mu he bi chi jiao mu wai ,8zhu jie kang jiao mu jun neng gou da fu du de jiang di li guo shi cai hou qing mei bing de fa sheng ;tong shi ,mie huo de jiao mu jun ti ji jiao mu xi bao bi jun neng you dao li guo shi dui qing mei bing de kang xing ,ji zhong yi hai xiang gong dong bao jiao mu (Rhodosporidium paludigenum)de mie huo jun ti he xi bao bi de xiao guo zui jia 。dui jiao mu xi bao bi zu fen fen xi biao ming ,bu tong lai yuan de tong chong jiao mu xi bao bi zu cheng shang ju you gao du xiang shi xing ,dan bu tong chong jie kang jiao mu de xi bao bi zu cheng shang cha yi jiao da 。jin yi bu dui xi bao bi duo tang zu fen de fen xi biao ming ,lai yuan yu niang jiu jiao mu he gong dong bao jiao mu xi bao bi de ji ding zhi 、β-1,3-D-pu ju tang he β-1,6-D-pu ju tang jun neng you dao li guo shi chan sheng dui qing mei bing de kang xing ,er gan lou tang dan bai dui li guo shi qing mei bing de kang xing mei you xian zhe de ying xiang 。(2)niang jiu jiao mu xi bao bi ji ding zhi de jie gou jian ding ji dui fan jia guo shi kang xing de you dao zuo yong cai yong wu shi nian du ji 、gao xiao ye xiang se pu (HPLC)、fu li xie gong wai guang pu (FTIR)he he ci gong zhen (NMR)dui niang jiu jiao mu xi bao bi ji ding zhi de jie gou jin hang le biao zheng ,bing tan jiu le ji dui fan jia hui mei bing de fang zhi xiao li ji xiang guan ji li 。yan jiu jie guo biao ming ,jiao mu xi bao bi ji ding zhi you chan tang N-yi xian an ji pu tao tang zu cheng ,fen zi liang wei 4.68×10~5 Dalton,tuo yi xian du wei 45.83%。ji ding zhi bu neng zhi jie yi zhi hui pu tao bao (Botrytis cinerea)de sheng chang ,dan ke yi xian zhe you dao fan jia guo shi dui you B.cinereayin qi de hui mei bing de di kang neng li ,ju you dao xiao guo yu chu li nong du he you dao shi jian mi qie xiang guan 。jin yi bu de fen xi jie guo biao ming ,jiao mu xi bao bi ji ding zhi you dao le guo shi zu zhi zhong huo xing yang (ROS)de ji lei he pian zhi zhi de chen ji ;tong shi di gao le guo shi fang yu xiang guan mei (SOD,CAT,POD,PAL,GLUhe CHI)huo xing de zeng jia ji ji xiang ying de ji yin de shang diao ;er ju jiao mu xi bao bi ji ding zhi chu li hai ji huo le shui yang suan xin hao tong lu guan jian ji yin (ICS,NPR1,TGA1a,TGA2,PR1b1he PR5)de shang diao biao da 。(3)niang jiu jiao mu xi bao bi β-1,3-D-pu ju tang de jie gou jian ding ji sheng fang xiao li ping jia tong guo wu shi nian du ji 、HPLC、FTIRhe NMRdeng ji shu shou duan dui β-1,3-D-pu ju tang de fen zi liang (M_w)、chan tang zu cheng 、te zheng xing guan neng tuan he tan qing xin hao jin hang le biao zheng 。jie guo biao ming ,fen li de dao de β-1,3-D-pu ju tang yang pin you chan tang pu tao tang zu cheng ,fen zi liang wei 165 kDa,pu tao tang fen zi jian tong guo β-1,3tang gan jian lian jie 。zai you dao guo shi kang xing fang mian ,β-1,3-D-pu ju tang bu neng zhi jie ying xiang kuo zhan qing mei (Penicillium expansum)bao zi zai ti wai he guo shi shang kou chu de meng fa ,er β-1,3-D-pu ju tang you dao chu li xian zhe yi zhi le guo shi shang kou chu P.expansumbao zi de meng fa 。sao miao dian jing (SEM)guan cha β-1,3-D-pu ju tang chu li dui li guo shi shang kou zu zhi chao wei jie gou ying xiang de jie guo biao ming ,β-1,3-D-pu ju tang ke yi zai xi bao zu zhi shui ping shang di gao dui P.expansumde kang xing 。RT-qPCRjie guo biao ming ,β-1,3-D-pu ju tang chu li xian zhe di gao le guo shi shang kou zhou wei zu zhi zhong PR1、GLU、endoGLU9、CHI3、CHI4、endoCHI、PR4、PR5he PALji yin de shang diao biao da ,jin er jiang di le li guo shi qing mei bing fu lan de fa bing lv he bing ban zhi jing 。(4)jiao mu xi bao bi pu ju tang ge cheng xiang guan ji yin Rho1de wei chuan zhuai hua ji ji sheng fang xiao li ping jia li yong te yi xing yin wu cong niang jiu jiao mu ji yin zu DNAzhong kuo zeng de dao mu de ji yin Rho1quan chang xu lie ,ke long zhi zai ti pYES6/CThou ,tong guo dian zhuai hua ji shu dao ru zhi niang jiu jiao mu zhong ,ce xu jian ding hou cheng gong de dao guo biao da niang jiu jiao mu jun zhu SC/Rho1。RT-qPCRjie guo xian shi ,you dao pei yang 12 hhou chong zu zi SC/Rho1zhong Rho1ji yin de biao da liang jiao ye sheng xing jun zhu shang diao le 107.89bei 。tong shi ,chong zu zi SC/Rho1xi bao bi zhong pu ju tang han liang zeng jia le 9.26%。you dao kang xing jie guo biao ming ,you dao pei yang hou de jiao mu xi bao bi xian shi chu dui li guo shi geng jiang de you dao xiao guo 。(5)jiao mu xi bao bi β-1,3-D-pu ju tang dui you dao li guo shi qing mei bing kang xing de zhuai lu zu xue fen xi dui β-1,3-D-pu ju tang he bing yuan jun chu li hou de li guo shi zu zhi jin hang zhuai lu zu xue fen xi 。jie guo biao ming ,jing niang jiu jiao mu xi bao bi β-1,3-D-pu ju tang yu bing yuan jun chu li hou de li guo rou zu zhi xiang bi yu dui zhao zu you 5984ge ji yin shang diao biao da ,8769ge ji yin xia diao biao da 。β-1,3-D-pu ju tang ke neng tong guo ji huo le li guo shi xi bao zhong mo shi fen zi shi bie shou ti FLS2、CNGCshe gai li zi xin hao tong dao ,yin fa le xia you de ji su xin hao sheng chang su 、mo li suan 、chi mei su he yi xi de chuan dao ,dao zhi fang yu xiang guan zhuai lu yin zi (MYC2、PTI5、PTI6、WRKY29he WRKY33)de biao da ,jin er chan sheng yi ji lie de fang yu fan ying ,bao gua fang yu xiang guan ji yin POD、PAL、HSP90he PR1de bian hua 、an ji suan de sheng wu ge cheng he dai xie ,yi ji ci ji dai xie tong lu de ji huo ,cong er yi zhi le li guo shi qing mei bing de fa sheng he fa zhan 。
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论文作者分别是来自浙江大学的孙翠，发表于刊物浙江大学2019-07-16论文，是一篇关于酵母细胞壁论文,几丁质论文,葡聚糖论文,扩展青霉论文,灰葡萄孢论文,浙江大学2019-07-16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，资料来自浙江大学2019-07-16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。