發(fā)布時(shí)間：2018-06-03 17:26

  本文選題：青枯菌 + 效應(yīng)蛋白　； 參考：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：由青枯菌侵染導(dǎo)致的青枯病是植物的一個(gè)重要的細(xì)菌性病害。青枯菌三型分泌系統(tǒng)效應(yīng)蛋白是其致病的重要因子。青枯菌的三型分泌系統(tǒng)效應(yīng)蛋白眾多,但是關(guān)于其致病的分子機(jī)制目前還了解的比較少。為了進(jìn)一步快速切入青枯菌效應(yīng)蛋白的毒力功能研究,本實(shí)驗(yàn)選用了瞬時(shí)表達(dá)體系對青枯菌的效應(yīng)蛋白在煙草上進(jìn)行了細(xì)胞死亡的篩選,并初步鑒定其介導(dǎo)的植物免疫途徑,同時(shí)篩選了其中能夠干擾植物ETI及PTI免疫途徑的效應(yīng)蛋白。本研究將為青枯菌效應(yīng)蛋白的毒力功能的深入探索奠定前期基礎(chǔ),為進(jìn)一步發(fā)掘抗青枯菌基因做好鋪墊。結(jié)果如下:1.利用青枯菌UW551對本氏煙、3種栽培種煙草(N.tabacum D101、N.tabacum云煙87、N.tabacum K326)、及4種野生型煙草(N.langsdorffii、N.repanda、N.alata、N.goodspeedii)進(jìn)行根部及葉片接種,結(jié)果表明,青枯菌UW551無法導(dǎo)致以上幾種煙草發(fā)病萎蔫,但能夠致使葉片出現(xiàn)細(xì)胞死亡。UW551的T3SS突變體△hrpB在煙草葉片上并無細(xì)胞死亡反應(yīng)。說明UW551產(chǎn)生的細(xì)胞死亡現(xiàn)象是由T3SS介導(dǎo)的。2.從青枯菌UW551中克隆了47個(gè)效應(yīng)子,并利用馬鈴薯病毒雙元表達(dá)載體pGR106成功構(gòu)建了其中42個(gè)效應(yīng)子基因的瞬時(shí)表達(dá)載體。3.利用農(nóng)桿菌介導(dǎo)的瞬時(shí)表達(dá)系統(tǒng)在本氏煙及7種煙草上對這些效應(yīng)子進(jìn)行鑒定發(fā)現(xiàn),效應(yīng)子Rip5可在所有的測試植物上誘導(dǎo)葉片組織的細(xì)胞死亡;Rip6可在本氏煙及4種野生型煙草上誘導(dǎo)葉片組織的細(xì)胞死亡;另外,Rip25、Rip26、Rip32、Rip 40、Rip 55、Rip 56、Hyp9可引起本氏煙葉片組織的不同程度的細(xì)胞死亡。4.對Rip5、Rip6、Rip25、Rip26、Rip55、Rip56在本氏煙上的免疫途徑中的功能研究,分別沉默植物免疫途徑的關(guān)鍵基因NbBAK1、NbNDR1、NbEDS1、NbSGT1、NbWIPK、NbCoi1,發(fā)現(xiàn)Rip26在NbBAK1沉默植株中誘導(dǎo)的細(xì)胞死亡增強(qiáng),而在NbNDR1的沉默植株中則明顯減弱。5.在UW551的效應(yīng)子對本氏煙葉片ETI與PTI介導(dǎo)細(xì)胞死亡的抑制分析中發(fā)現(xiàn),有7個(gè)能夠完全抑制INF1激發(fā)的HR,分別是Rip6、Rip10、Rip13、Rip28、Rip30、Rip32、Rip47、Hyp11;有9個(gè)能夠抑制PiCRN2激發(fā)的HR,分別是Rip6、Rip10、Rip13、Rip30、Rip31、Rip32、Rip36、Rip53、Hyp11;Rip6能夠部分抑制Avr3a/R3a、Avr4/Cf4激發(fā)的HR。青枯菌UW551的部分效應(yīng)子對PCD具有相同的抑制作用,其在病原菌侵染中可能抑制PTI、ETI的免疫反應(yīng)。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S435.72

【參考文獻(xiàn)】

相關(guān)期刊論文 前9條

1 楊銀;高志勇;;植物抗病機(jī)制的研究進(jìn)展[J];科教導(dǎo)刊(中旬刊);2016年09期

2 張勇;李牧原;羅鋒;;青枯菌三型分泌系統(tǒng)研究進(jìn)展[J];微生物學(xué)報(bào);2015年06期

3 隋新霞;郭棟;尤升波;;植物非寄主抗性的遺傳與機(jī)理研究進(jìn)展[J];山東農(nóng)業(yè)科學(xué);2014年08期

4 閆慧芳;毛培勝;夏方山;;植物抗氧化劑谷胱甘肽研究進(jìn)展[J];草地學(xué)報(bào);2013年03期

5 孔瓊;楊紅玉;王云月;霍達(dá);胡海林;劉振華;秦麗娟;;植物與病原物互作中的細(xì)胞程序化死亡[J];植物保護(hù);2012年06期

6 陳英;譚碧s，

本文編號：1973596