本文關鍵詞：南方根結(jié)線蟲MiMsp40效應子與寄主STZ互作調(diào)控免疫的機理研究　出處：《中國農(nóng)業(yè)大學》2017年博士論文　論文類型：學位論文

  更多相關文章： 南方根結(jié)線蟲 MiMsp40效應因子 免疫反應 STZ轉(zhuǎn)錄抑制子 互作

【摘要】：南方根結(jié)線蟲是一種重要的植物寄生線蟲,營固著性、活體內(nèi)寄生,寄主范圍廣泛,造成巨大的經(jīng)濟損失,嚴重威脅農(nóng)業(yè)生產(chǎn)安全,目前還缺少安全有效的防治辦法。深入研究線蟲效應因子的致病機理是目前相關研究工作關注的熱點。本研究在實驗室已有研究結(jié)果的基礎上,進一步深入研究了南方根結(jié)線蟲效應因子MiMsp40的功能,結(jié)果總結(jié)如下:實驗結(jié)果表明,MiMsp40具有廣泛抑制植物免疫相關反應的作用。MiMsp40在擬南芥中的異位表達能夠抑制免疫反應相關基因FRK1、WRKY29、WRKY33和CYP81F2的表達,并抑制擬南芥的胼胝質(zhì)沉積。MiMsp40在煙草葉片中瞬時表達,能夠抑制由BAX、NPK1和MKK1激發(fā)的細胞壞死反應,還能抑制ETI激發(fā)子R3a/Avr3a引起的細胞壞死反應。對MiMsp40轉(zhuǎn)基因擬南芥的轉(zhuǎn)錄組測序分析顯示,MiMsp40轉(zhuǎn)基因擬南芥的茉莉酸生物合成關鍵基因LOX3、AOS、AOC和OPR3,茉莉酸信號傳導通路關鍵基因JAZ家族和MYC2等的表達均受到抑制。與Col-0型相比,兩個MiMsp40轉(zhuǎn)基因擬南芥中的茉莉酸相對濃度顯著下降(P0.05),分別為20%±9%和36%±11%。對MiMsp40轉(zhuǎn)基因擬南芥進行茉莉酸回補后,植株對線蟲的敏感性消失,證明MiMsp40調(diào)控了擬南芥的茉莉酸生物合成通路。在分析轉(zhuǎn)錄組測序結(jié)果的基礎上,發(fā)現(xiàn)了一個調(diào)控茉莉酸生物合成通路關鍵基因LOX3的轉(zhuǎn)錄因子STZ。通過免疫共沉淀和雙分子熒光互補分析,證明MiMsp40與STZ在植物細胞核中特異互作。通過對STZ調(diào)控LOX3啟動子序列的分析,證明了 STZ是LOX3的轉(zhuǎn)錄抑制子。在擬南芥中突變STZ會使上述四個茉莉酸合成關鍵基因的表達上調(diào)。與Col-0型相比,STZ突變體擬南芥在接種線蟲后,產(chǎn)生的根結(jié)數(shù)和雌成蟲數(shù)分別下降了 42%和47%,植株對線蟲表現(xiàn)出抗性。進一步分析MiMsp40與STZ的互作發(fā)現(xiàn),MiMsp40可以促進植物細胞內(nèi)STZ蛋白的積累。因此,推測MiMsp40是通過促進STZ積累,負調(diào)控LOX3的轉(zhuǎn)錄,減少植物茉莉酸類激素的合成,從而達到促進線蟲寄生致病的目的。對前人通過酵母雙雜交篩選得到的另一個MiMsp40潛在互作蛋白Hs1pro-1及其同源蛋白Hs1pro-2進行了分析。免疫共沉淀和雙分子熒光互補分析證明MiMsp40與Hs1pro-1和Hs1pro-2分別互作。對Hs1pro-i突變體擬南芥進行線蟲敏感性、細菌性病原物丁香假單胞菌敏感性及免疫相關基因表達等分析表明,Hs1pro-1突變體擬南芥對線蟲和丁香假單胞菌均表現(xiàn)抗性,植物的免疫相關基因FRK1、PR1和CYP81F2顯著上調(diào),證明了Hs1pro-1不是傳統(tǒng)認為的抗性基因,拓展了對Hs1pro-1基因功能的認識,說明Hs1pro-1/2在植物與病原物的互作過程中可能具有更加復雜的作用。
[Abstract]:Root-knot nematode is an important plant parasitic nematodes, camp fixed, living parasitic, host a wide range, causing huge economic losses, serious threats to the safety of agricultural production. At present, there is a lack of safe and effective control methods. Further study on the pathogenic mechanism of nematode effect factors is the focus of relevant research work. This study is based on the results of laboratory research. The function of the effect factor MiMsp40 of the southern root knot nematode was further studied. The results were summarized as follows: the experimental results showed that. The heterotopic expression of MiMsp40 in Arabidopsis thaliana can inhibit the immune response related gene FRK1 and WRKY29. The expression of WRKY33 and CYP81F2 and inhibition of transient expression of callosum deposition. MiMsp40 in tobacco leaves could be inhibited by BAX. Cell necrosis induced by NPK1 and MKK1. It also inhibited the cell necrosis induced by ETI exciter R3a / Avr3a. The transcriptome sequencing of MiMsp40 transgenic Arabidopsis thaliana showed that. The key genes of jasmonic acid biosynthesis in Arabidopsis thaliana transgenic with MiMsp40 are LOX3AOC and OPR3. The expression of JAZ family and MYC2, the key genes of jasmonic acid signal transduction pathway, were inhibited, compared with Col-0 type. The relative concentration of jasmonic acid in two MiMsp40 transgenic Arabidopsis thaliana decreased significantly (P 0.05). The MiMsp40 transgenic Arabidopsis thaliana was supplemented with jasmonic acid, and the plant sensitivity to nematodes disappeared. It is proved that MiMsp40 regulates the biosynthesis pathway of Jasmonic acid in Arabidopsis thaliana. A transcription factor, LOX3, which regulates the biosynthesis pathway of jasmonic acid, was identified by immunoprecipitation and bimolecular fluorescence complementary analysis. The specific interaction between MiMsp40 and STZ in plant nuclei was demonstrated. The sequence of LOX3 promoter regulated by STZ was analyzed. It is proved that STZ is the transcription suppressor of LOX3. The mutated STZ in Arabidopsis can up-regulate the expression of the four key genes of jasmonic acid biosynthesis, compared with the Col-0 type. The root knot number and female adult number of STZ mutant Arabidopsis decreased by 42% and 47% respectively after inoculation with nematodes. The plant showed resistance to nematodes. Further analysis of the interaction between MiMsp40 and STZ found that MMP 40 could promote the accumulation of STZ protein in plant cells. It is suggested that MiMsp40 can reduce the synthesis of plant jasmonates by promoting the accumulation of STZ and negatively regulating the transcription of LOX3. In order to promote the parasitic pathogenicity of nematodes, another MiMsp40 potential interaction protein Hs1pro-1 and its homologous protein Hs1pro-2 were obtained by yeast two-hybrid screening. Immunocoprecipitation and bimolecular fluorescence complementary analysis showed that MiMsp40 interacted with Hs1pro-1 and Hs1pro-2, respectively. The line of Hs1pro-i mutant Arabidopsis thaliana. Worm sensitivity. The analysis of susceptibility and immune-related gene expression showed that Arabidopsis thaliana, a mutant of Hs1pro-1, was resistant to both nematode and eugenomonas. The immune-related genes FRK1 PR1 and CYP81F2 were significantly up-regulated in plants, which suggested that Hs1pro-1 was not a traditional resistant gene. The understanding of the function of Hs1pro-1 gene is extended, which indicates that Hs1pro-1/2 may play a more complicated role in the interaction between plant and pathogen.
【學位授予單位】：中國農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S432.45

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