南方根结线虫MiMsp40效应子与寄主STZ互作调控免疫的机理研究

发布时间：2018-01-16 23:02

本文关键词：南方根结线虫MiMsp40效应子与寄主STZ互作调控免疫的机理研究　出处：《中国农业大学》2017年博士论文　论文类型：学位论文

【摘要】：南方根结线虫是一种重要的植物寄生线虫,营固着性、活体内寄生,寄主范围广泛,造成巨大的经济损失,严重威胁农业生产安全,目前还缺少安全有效的防治办法。深入研究线虫效应因子的致病机理是目前相关研究工作关注的热点。本研究在实验室已有研究结果的基础上,进一步深入研究了南方根结线虫效应因子MiMsp40的功能,结果总结如下:实验结果表明,MiMsp40具有广泛抑制植物免疫相关反应的作用。MiMsp40在拟南芥中的异位表达能够抑制免疫反应相关基因FRK1、WRKY29、WRKY33和CYP81F2的表达,并抑制拟南芥的胼胝质沉积。MiMsp40在烟草叶片中瞬时表达,能够抑制由BAX、NPK1和MKK1激发的细胞坏死反应,还能抑制ETI激发子R3a/Avr3a引起的细胞坏死反应。对MiMsp40转基因拟南芥的转录组测序分析显示,MiMsp40转基因拟南芥的茉莉酸生物合成关键基因LOX3、AOS、AOC和OPR3,茉莉酸信号传导通路关键基因JAZ家族和MYC2等的表达均受到抑制。与Col-0型相比,两个MiMsp40转基因拟南芥中的茉莉酸相对浓度显著下降(P0.05),分别为20%±9%和36%±11%。对MiMsp40转基因拟南芥进行茉莉酸回补后,植株对线虫的敏感性消失,证明MiMsp40调控了拟南芥的茉莉酸生物合成通路。在分析转录组测序结果的基础上,发现了一个调控茉莉酸生物合成通路关键基因LOX3的转录因子STZ。通过免疫共沉淀和双分子荧光互补分析,证明MiMsp40与STZ在植物细胞核中特异互作。通过对STZ调控LOX3启动子序列的分析,证明了 STZ是LOX3的转录抑制子。在拟南芥中突变STZ会使上述四个茉莉酸合成关键基因的表达上调。与Col-0型相比,STZ突变体拟南芥在接种线虫后,产生的根结数和雌成虫数分别下降了 42%和47%,植株对线虫表现出抗性。进一步分析MiMsp40与STZ的互作发现,MiMsp40可以促进植物细胞内STZ蛋白的积累。因此,推测MiMsp40是通过促进STZ积累,负调控LOX3的转录,减少植物茉莉酸类激素的合成,从而达到促进线虫寄生致病的目的。对前人通过酵母双杂交筛选得到的另一个MiMsp40潜在互作蛋白Hs1pro-1及其同源蛋白Hs1pro-2进行了分析。免疫共沉淀和双分子荧光互补分析证明MiMsp40与Hs1pro-1和Hs1pro-2分别互作。对Hs1pro-i突变体拟南芥进行线虫敏感性、细菌性病原物丁香假单胞菌敏感性及免疫相关基因表达等分析表明,Hs1pro-1突变体拟南芥对线虫和丁香假单胞菌均表现抗性,植物的免疫相关基因FRK1、PR1和CYP81F2显著上调,证明了Hs1pro-1不是传统认为的抗性基因,拓展了对Hs1pro-1基因功能的认识,说明Hs1pro-1/2在植物与病原物的互作过程中可能具有更加复杂的作用。
[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.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S432.45

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