發(fā)布時(shí)間：2018-05-14 17:29

  本文選題：茉莉酸途徑 + 油菜素甾醇途徑�。� 參考：《浙江大學(xué)》2017年博士論文

【摘要】：植物激素在植物免疫反應(yīng)過(guò)程中具有重要作用,然而植物激素與水稻黑條矮縮病毒(Rice black-streaked dwarf virus,RBSDV)互作的報(bào)道卻很少。本研究對(duì)健康葉片和感染RBSDV病葉進(jìn)行高通量測(cè)序分析病毒侵染后寄主基因組表達(dá)譜的變化,篩選與病毒侵染相關(guān)的激素途徑。采用超高效液相-三重四級(jí)桿質(zhì)譜法(UPLC-MS/MS)檢測(cè)病毒侵染后寄主內(nèi)源性激素濃度。運(yùn)用外源性激素和水稻突變體研究各激素途徑在RBSDV侵染水稻過(guò)程中的作用。具體結(jié)果如下:1.高通量測(cè)序結(jié)果表明在RBSDV侵染后,很多寄主防御和應(yīng)激相關(guān)基因上調(diào)表達(dá)。Real time quantitative PCR(RT-qPCR)對(duì)部分基因進(jìn)行驗(yàn)證,結(jié)果顯示被檢測(cè)的10個(gè)PR基因和11個(gè)應(yīng)激相關(guān)轉(zhuǎn)錄因子在病毒侵染30和60d時(shí)都顯著性上調(diào)表達(dá)。2.高通量測(cè)序和RT-qPCR結(jié)果顯示茉莉酸(Jasmonic acid,JA)合成途徑相關(guān)基因在病毒侵染后顯著性上調(diào)表達(dá)。JA應(yīng)答標(biāo)志性基因JiOsPR10在病毒侵染30和60 d時(shí)都顯著性上調(diào)表達(dá)。UPLC-MS/MS檢測(cè)結(jié)果表明感病水稻內(nèi)源性JA濃度升高,為健康水稻的4.1倍。外源性MeJA處理水稻后RBSDV發(fā)病率顯著性低于對(duì)照(0.1%Triton X-100)處理的水稻。JA受體OsCOI1-RNAi突變體coi1-13的病毒發(fā)病率及感病植株中RBSDV外殼蛋白mRNA的表達(dá)量都顯著性高于野生型對(duì)照組。這些結(jié)果表明JA途徑在水稻抵抗RBSDV侵染過(guò)程中發(fā)揮正調(diào)控作用。3.高通量測(cè)序結(jié)果分析發(fā)現(xiàn)油菜素甾醇(brassinosteroids,BR)合成途徑相關(guān)基因在RBSDV侵染后下調(diào)表達(dá)。RT-qPCR實(shí)驗(yàn)結(jié)果顯示BR合成途徑和信號(hào)途徑相關(guān)基因在病毒侵染30和60 d時(shí)顯著性下調(diào)表達(dá)。UPLC-MS/MS檢測(cè)結(jié)果表明感病水稻內(nèi)源性油菜素甾酮(castasterone,CS)濃度顯著性下降,為健康水稻的0.45倍。外源性表油菜素內(nèi)酯(epibrassinolide,BL)處理水稻后RBSDV發(fā)病率顯著性高于對(duì)照(0.1%Triton X-100)處理的水稻,而B(niǎo)R途徑抑制劑蕓苔素唑(brassinazole,BRZ)處理水稻后其病毒發(fā)病率顯著性低于對(duì)照處理的水稻。OsGSK2過(guò)表達(dá)突變體Go的病毒發(fā)病率及感病植株中RBSDV外殼蛋白mRNA的表達(dá)量都顯著性低于對(duì)照植株中花11(Zh11)。這些結(jié)果表明激活BR途徑有利于RBSDV侵染水稻。4.MeJA和BL混合液處理組RBSDV發(fā)病率顯著性低于對(duì)照組。MeJA處理野生型水稻6和12h時(shí),JA途徑相關(guān)基因上調(diào)表達(dá)而B(niǎo)R途徑相關(guān)基因下調(diào)表達(dá)。這表明水稻中激活JA途徑后能夠抑制BR合成和信號(hào)途徑相關(guān)基因表達(dá)。在MeJA處理12 h時(shí),對(duì)照日本晴的BR合成和信號(hào)途徑相關(guān)基因顯著性下調(diào)表達(dá),而突變體coi1-13未發(fā)生顯著性差異變化。這些結(jié)果表明水稻中激活后的JA途徑抑制BR合成和信號(hào)途徑相關(guān)基因表達(dá)受到OsCO/1受體的調(diào)控。綜上所述,本研究結(jié)果表明防御和應(yīng)激相關(guān)基因及JA途徑相關(guān)基因在病毒侵染后上調(diào)表達(dá)而B(niǎo)R途徑相關(guān)基因下調(diào)表達(dá)。在RBSDV侵染水稻過(guò)程中,JA途徑是抗病性途徑而B(niǎo)R途徑是感病性途徑。受體OsC9/1調(diào)控JA途徑誘導(dǎo)的寄主抗病性抑制BR途徑誘導(dǎo)的寄主感病性。
[Abstract]:Plant hormones play an important role in plant immune response, but there are few reports on the interaction of plant hormones with Rice Rice black-streaked dwarf virus (RBSDV). In this study, the high throughput sequencing of healthy leaves and infected leaves with RBSDV disease was carried out to analyze the changes of host genome expression profiles after virus infection, and to screen the hormone pathway related to virus infection. UPLC-MS / MS was used to detect the concentration of host endogenous hormones after virus infection. Exogenous hormones and rice mutants were used to study the role of hormone pathway in the process of RBSDV infection in rice. The results are as follows: 1. The results of high-throughput sequencing showed that many host defense and stress-related genes upregulated the expression of .Real time quantitative PCRRT-qPCRR, and some of the genes were verified by RT-PCR. The results showed that 10 PR genes and 11 stress-related transcription factors were significantly up-regulated at 30 and 60 days after infection. High throughput sequencing and RT-qPCR results showed that the genes associated with Jasmonic acid jar) biosynthesis pathway significantly upregulated the expression of .JA response marker gene JiOsPR10 at 30 and 60 days after infection with the virus. The concentration of endogenous JA in susceptible rice increased. 4. 1 times as much as healthy rice. The incidence of RBSDV in rice treated with exogenous MeJA was significantly lower than that in rice. JA receptor OsCOI1-RNAi mutant coi1-13 treated with 0.1 Triton X-100) and the expression of RBSDV coat protein mRNA in susceptible plants was significantly higher than that in wild type control. These results suggest that the JA pathway plays a positive regulatory role in the process of resistance to RBSDV infection in rice. The results of high-throughput sequencing showed that BrassinosteroidsBR-associated genes down-regulated the expression of BrassinosteroidsBR-related genes after RBSDV infection. The results of RT-qPCR showed that Br biosynthesis pathway and signaling pathway genes were significantly down-regulated at 30 and 60 days after infection with the virus. The results of UPLC-MS / MS analysis showed that the concentration of endogenous rapesone castasterone in susceptible rice decreased significantly. 0.45 times as much as healthy rice. The incidence of RBSDV in rice treated with exogenous epibrassinolide was significantly higher than that in rice treated with 0.1 Triton X-100. However, the virus incidence of Brassinazoleine BrZ treated with Br pathway inhibitor was significantly lower than that of rice. OsGSK2 overexpression mutants go, and the expression of RBSDV coat protein mRNA in susceptible plants was significantly lower than that of control treatment. In the control plant, the flower was 11. These results indicated that activation of Br pathway was beneficial to the incidence of RBSDV in RBSDV infected rice. 4. Meja and BL mixed solution treatment group was significantly lower than that of control group at 6 and 12 hours after treatment with .MeJA treatment, the expression of genes related to JA pathway in wild type rice was up-regulated, but the expression of Br pathway related genes was down-regulated. This indicated that JA pathway activation could inhibit Br synthesis and signal pathway related gene expression in rice. After 12 h of MeJA treatment, Br synthesis and signal pathway related genes were significantly down-regulated in the control group, while the mutant coi1-13 had no significant difference. These results suggest that the activated JA pathway inhibits Br synthesis and signaling pathway related gene expression in rice by OsCO/1 receptor. In conclusion, the results showed that defense and stress related genes and JA pathway related genes were up-regulated and Br pathway genes were down-regulated after virus infection. In the process of RBSDV infection, the JA pathway is a disease resistance pathway and the Br pathway is a susceptible pathway. The receptor OsC9/1 regulates the resistance of hosts induced by JA pathway and inhibits the resistance of hosts induced by Br pathway.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S435.111.4

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