烟草上基于瞬时表达系统的青枯菌UW551的毒力效应蛋白的筛选

发布时间：2018-06-03 17:26

本文选题：青枯菌 + 效应蛋白　；参考：《华中农业大学》2017年硕士论文

【摘要】：由青枯菌侵染导致的青枯病是植物的一个重要的细菌性病害。青枯菌三型分泌系统效应蛋白是其致病的重要因子。青枯菌的三型分泌系统效应蛋白众多,但是关于其致病的分子机制目前还了解的比较少。为了进一步快速切入青枯菌效应蛋白的毒力功能研究,本实验选用了瞬时表达体系对青枯菌的效应蛋白在烟草上进行了细胞死亡的筛选,并初步鉴定其介导的植物免疫途径,同时筛选了其中能够干扰植物ETI及PTI免疫途径的效应蛋白。本研究将为青枯菌效应蛋白的毒力功能的深入探索奠定前期基础,为进一步发掘抗青枯菌基因做好铺垫。结果如下:1.利用青枯菌UW551对本氏烟、3种栽培种烟草(N.tabacum D101、N.tabacum云烟87、N.tabacum K326)、及4种野生型烟草(N.langsdorffii、N.repanda、N.alata、N.goodspeedii)进行根部及叶片接种,结果表明,青枯菌UW551无法导致以上几种烟草发病萎蔫,但能够致使叶片出现细胞死亡。UW551的T3SS突变体△hrpB在烟草叶片上并无细胞死亡反应。说明UW551产生的细胞死亡现象是由T3SS介导的。2.从青枯菌UW551中克隆了47个效应子,并利用马铃薯病毒双元表达载体pGR106成功构建了其中42个效应子基因的瞬时表达载体。3.利用农杆菌介导的瞬时表达系统在本氏烟及7种烟草上对这些效应子进行鉴定发现,效应子Rip5可在所有的测试植物上诱导叶片组织的细胞死亡;Rip6可在本氏烟及4种野生型烟草上诱导叶片组织的细胞死亡;另外,Rip25、Rip26、Rip32、Rip 40、Rip 55、Rip 56、Hyp9可引起本氏烟叶片组织的不同程度的细胞死亡。4.对Rip5、Rip6、Rip25、Rip26、Rip55、Rip56在本氏烟上的免疫途径中的功能研究,分别沉默植物免疫途径的关键基因NbBAK1、NbNDR1、NbEDS1、NbSGT1、NbWIPK、NbCoi1,发现Rip26在NbBAK1沉默植株中诱导的细胞死亡增强,而在NbNDR1的沉默植株中则明显减弱。5.在UW551的效应子对本氏烟叶片ETI与PTI介导细胞死亡的抑制分析中发现,有7个能够完全抑制INF1激发的HR,分别是Rip6、Rip10、Rip13、Rip28、Rip30、Rip32、Rip47、Hyp11;有9个能够抑制PiCRN2激发的HR,分别是Rip6、Rip10、Rip13、Rip30、Rip31、Rip32、Rip36、Rip53、Hyp11;Rip6能够部分抑制Avr3a/R3a、Avr4/Cf4激发的HR。青枯菌UW551的部分效应子对PCD具有相同的抑制作用,其在病原菌侵染中可能抑制PTI、ETI的免疫反应。
[Abstract]:Bacterial wilt caused by bacterial wilt infection is an important bacterial disease in plants. The secretory system effector protein of the three types of bacterial wilt is an important factor of its pathogenesis. There are a lot of secretory system effector proteins of bacterial wilt, but little is known about the molecular mechanism of its pathogenicity. In order to further study the virulence function of bacterial Wilt effector protein, the transient expression system was used to screen the effector protein of Bacillus solanacearum on tobacco, and its mediated plant immune pathway was preliminarily identified. At the same time, the effector proteins which can interfere with the immune pathway of ETI and PTI in plants were screened. This study will lay a foundation for the further exploration of virulence function of bacterial effector protein and pave the way for further exploring the genes of bacterial wilt resistance. The result is as follows: 1. UW551 was used to inoculate N.tabacum D101N. Tabacum K326 and N. langsdorffiiae N.alatagoodN.speedii. the results showed that UW551 could not cause wilting of tobacco. However, the T3SS mutant hrpB, which could cause cell death in leaves, did not respond to cell death in tobacco leaves. The results showed that the cell death caused by UW551 was mediated by T3SS. 47 effectors were cloned from bacterial wilt UW551 and the transient expression vector of 42 effector genes was constructed by using potato virus bivariate expression vector pGR106. The transient expression system mediated by Agrobacterium tumefaciens was used to identify these effectors in Bentworth tobacco and 7 tobacco species. The effector Rip5 could induce cell death of leaf tissue in all tested plants. Rip6 could induce cell death of leaf tissue on Bentworth tobacco and four wild tobacco species. In addition, Rip25, Rip26, Rip32, Rip40, Rip50, Rip56, Hyp9 can cause different degrees of cell death. The function of Rip56 rip25 rip26 rip56 in the immune pathway of Bensi tobacco was studied. NbBAK1NbNDR1NbNDR1NbNDR1NbNDR1NbNDR1NbSGT1NbWIPKNbCoi1 was silenced respectively. It was found that the cell death induced by Rip26 in NbBAK1 silencing plants was enhanced, while that in NbNDR1 silencing plants was significantly decreased. In the analysis of the inhibitory effects of UW551 effectors on ETI and PTI induced cell death in tobacco leaves, we found that there were seven HRs that could completely inhibit the INF1 stimulation, respectively, which were Rip6A Rip10Rip13Rip13 Rip30 rip32Rip37 (Hyp11), and 9 HRPs that could inhibit PiCRN2 stimulation, respectively, Rip31Rip32Rip3ypH11P6 could partially inhibit the excitation of Avr3aR3p3ypH11Cf4. Some effectors of UW551 have the same inhibitory effect on PCD, which may inhibit the immune response of PCD in pathogen infection.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S435.72

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