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    蔷薇属植物响应蔷薇盘二孢侵染内参基因的筛选及茉莉酸相关基因表达分析

    Selection and validation of appropriate reference genes and expression analysis of jasmonic acid-related genes responding to Marssonina rosae in Rosa species and cultivars

      摘要
    • 摘要:
        目的  为研究蔷薇属植物响应黑斑病侵染的稳定表达的最适内参基因，解析茉莉酸在蔷薇属植物响应蔷薇盘二孢侵染过程中的作用。
        方法  以黑斑病病原菌蔷薇盘二孢侵染的6个蔷薇属种/品种不同时间的离体叶片为材料，利用qRT-PCR技术及geNorm、NormFinder、BestKeeper软件对9个候选内参基因（ACT、GAPDH、PP2A、Rcl2、SAND、TIP、TUA、TUB、UBC）的表达量进行测定和分析，利用筛选出的内参基因，对蔷薇属植物茉莉酸（JA）抗病途径相关基因（COI1、OPR3、MYC2、JAR1）的表达水平进行定量分析。
        结果  （1）UBC可作为6种蔷薇属植物共同适用的内参基因，可用于后续分析JA抗病途径相关基因的表达水平。（2）内源JA含量在6种蔷薇属植物响应蔷薇盘二孢侵染的过程中存在差异。在黑斑病高抗植物受侵染0 ~ 4 d间JA含量下调，4 ~ 8 d间上调。在黑斑病易感植物受侵染0 ~ 8 d间，内源JA含量呈下调趋势。（3）JA合成相关基因OPR3及JAR1表达量在受侵染初期表达量趋势存在差异。在除荷花蔷薇外的其余5种植物中，OPR3在侵染初期（0 ~ 0.5 d）表达下调，JAR1在侵染初期表达上调。OPR3和JAR1在侵染后期均表达上调，黑斑病易感材料的上调程度高于高抗材料。（4）JA信号传导相关基因COI1及MYC2在受侵染初期表达量趋势同样存在差异。COI1在黑斑病高抗材料受侵染初期上调表达，在黑斑病易感材料下调表达，MYC2在6种植物受侵染0 ~ 2 d中均下调表达。COI1及MYC2表达量在受侵染2 d后均上调表达，且在黑斑病易感植物中的上调程度大于黑斑病高抗材料。
        结论  与JA信号传导相关的MYC2、COI1在蔷薇属植物抵御黑斑病病菌入侵过程中发挥负调控作用，且由JA通路介导的抵御死体营养型病原菌的侵染在后期发挥了作用。

       

      Abstract:
        Objective  This paper aims to select the appropriate reference gene and analyze the role of jasmonic acid in Rosa species and cultivars responding to Marssonina rosae.
        Method  R. multiflora f. carnea, R. xanthina f. spontanea, R. glauca, R. rugosa, R. hybrida ‘Porcelina’ and R12-26 (hybrid of R. rugosa and R. hybrida ‘Porcelina’) were used as materials, qRT-PCR technology, geNorm, NormFinder, BestKeeper were used to evaluate the expression stability of nine candidate reference genes (ACT, GAPDH, PP2A, RCl2, SAND, TIP, TUA, TUB and UBC). The expression levels of jasmonic acid resistance pathway relatedgenes (COI1, OPR3, MYC2 and JAR1) in six Rosa species and cultivars were quantitatively analyzed.
        Result  (1) UBC was the common reference gene of six Rosa species and cultivars responding to M. rosae, and can be used for analyzing the related gene expression levels to JA resistance pathway. (2) There were differences in JA content among the six plants responding to M. rosae. In disease-resistant plants, JA concentration decreased at 0−4 d and increased at 4−8 d. In disease-susceptible plants, JA concentration decreased at 0−8 d and increased significantly at 8−10 d. (3) The expression levels of JA synthesis-related genes OPR3 and JAR1 were different at the early stage of infection. The expression level of OPR3 was down-regulated and JAR1 up-regulated in five plants except for R. multiflora f. carnea. OPR3 and JAR1 were all up-regulated in the late stage of infection, however the up-regulation degree in black-spot susceptible species was significantly higher than that of resistant species. (4) There were also differences in the expression levels of JA signaling related genes COI1 and MYC2 at the early stage of infection. The expression of COI1 was up-regulated in disease-resistant plants and down-regulated in disease-susceptible plants at the early stage of infection, MYC2 was down-regulated in 6 plants infected 0−2 d. COI1 and MYC2 were up-regulated after 2 d of infection, and the up-regulation degree in black-spot susceptible species was significantly higher than that of resistant species.
        Conclusion  MYC2 and COI1, which are related to JA signaling, play a negative regulatory role in the resistance of Rosa plants to the invasion of black spot pathogens, and the JA pathway mediated resistance to the invasion of dead vegetative pathogens plays a role in the later stage of infection.

       

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