茉莉酸途径抑制油菜素甾醇途径调控水稻免疫水稻黑条矮缩病毒的作用机制研究

发布时间：2018-05-14 17:29

本文选题：茉莉酸途径 + 油菜素甾醇途径　；参考：《浙江大学》2017年博士论文

【摘要】：植物激素在植物免疫反应过程中具有重要作用,然而植物激素与水稻黑条矮缩病毒(Rice black-streaked dwarf virus,RBSDV)互作的报道却很少。本研究对健康叶片和感染RBSDV病叶进行高通量测序分析病毒侵染后寄主基因组表达谱的变化,筛选与病毒侵染相关的激素途径。采用超高效液相-三重四级杆质谱法(UPLC-MS/MS)检测病毒侵染后寄主内源性激素浓度。运用外源性激素和水稻突变体研究各激素途径在RBSDV侵染水稻过程中的作用。具体结果如下:1.高通量测序结果表明在RBSDV侵染后,很多寄主防御和应激相关基因上调表达。Real time quantitative PCR(RT-qPCR)对部分基因进行验证,结果显示被检测的10个PR基因和11个应激相关转录因子在病毒侵染30和60d时都显著性上调表达。2.高通量测序和RT-qPCR结果显示茉莉酸(Jasmonic acid,JA)合成途径相关基因在病毒侵染后显著性上调表达。JA应答标志性基因JiOsPR10在病毒侵染30和60 d时都显著性上调表达。UPLC-MS/MS检测结果表明感病水稻内源性JA浓度升高,为健康水稻的4.1倍。外源性MeJA处理水稻后RBSDV发病率显著性低于对照(0.1%Triton X-100)处理的水稻。JA受体OsCOI1-RNAi突变体coi1-13的病毒发病率及感病植株中RBSDV外壳蛋白mRNA的表达量都显著性高于野生型对照组。这些结果表明JA途径在水稻抵抗RBSDV侵染过程中发挥正调控作用。3.高通量测序结果分析发现油菜素甾醇(brassinosteroids,BR)合成途径相关基因在RBSDV侵染后下调表达。RT-qPCR实验结果显示BR合成途径和信号途径相关基因在病毒侵染30和60 d时显著性下调表达。UPLC-MS/MS检测结果表明感病水稻内源性油菜素甾酮(castasterone,CS)浓度显著性下降,为健康水稻的0.45倍。外源性表油菜素内酯(epibrassinolide,BL)处理水稻后RBSDV发病率显著性高于对照(0.1%Triton X-100)处理的水稻,而BR途径抑制剂芸苔素唑(brassinazole,BRZ)处理水稻后其病毒发病率显著性低于对照处理的水稻。OsGSK2过表达突变体Go的病毒发病率及感病植株中RBSDV外壳蛋白mRNA的表达量都显著性低于对照植株中花11(Zh11)。这些结果表明激活BR途径有利于RBSDV侵染水稻。4.MeJA和BL混合液处理组RBSDV发病率显著性低于对照组。MeJA处理野生型水稻6和12h时,JA途径相关基因上调表达而BR途径相关基因下调表达。这表明水稻中激活JA途径后能够抑制BR合成和信号途径相关基因表达。在MeJA处理12 h时,对照日本晴的BR合成和信号途径相关基因显著性下调表达,而突变体coi1-13未发生显著性差异变化。这些结果表明水稻中激活后的JA途径抑制BR合成和信号途径相关基因表达受到OsCO/1受体的调控。综上所述,本研究结果表明防御和应激相关基因及JA途径相关基因在病毒侵染后上调表达而BR途径相关基因下调表达。在RBSDV侵染水稻过程中,JA途径是抗病性途径而BR途径是感病性途径。受体OsC9/1调控JA途径诱导的寄主抗病性抑制BR途径诱导的寄主感病性。
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S435.111.4

【参考文献】