Molecular mechanism and gene mining of passion fruit（Passiflora edulis） in response to infection of Phytophthora nicotianae
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摘要: 【目的】在转录水平上分析百香果响应烟草疫霉（Phytophthora nicotianae）侵染的表达模式，挖掘抗性相关基因并解析其作用机制，为利用分子手段进行百香果抗病性状筛选和抗病品种选育打下基础。【方法】以百香果主栽品种钦果9号为试材，对烟草疫霉侵染12、24、48、72和96 hpi钦果9号进行组织表型观察和生理指标测定；采用转录组测序（RNA-Seq）构建钦果9号响应烟草疫霉侵染的转录组，使用DESeq分析基因表达差异，以P<0.005、错误发现率（FDR）≤0.001和|log2 Ratio|≥1为标准筛选差异表达基因（DEGs）；通过BLAST2GO和KOBAS2等进行DEGs的GO功能注释分析和KEGG信号通路富集分析，明确钦果9号响应烟草疫霉侵染的主要通路和代谢途径；挖掘与植物抗病相关代谢通路的DEGs并利用MeV软件绘制基因表达热图，分析其在烟草疫霉侵染钦果9号过程中的动态变化趋势；实时荧光定量PCR验证基因表达量差异与转录组数据的一致性。【结果】烟草疫霉侵染钦果9号初期叶片无明显症状，侵染48 hpi叶片出现褪绿，随着侵染时间不断延长，病斑颜色变深并不断扩大呈明显棕色枯斑；从侵染12 hpi开始所有处理组钦果9号叶片中时超氧化物歧化酶（SOD）活性显著高于对照组（P<0.05），在24 hpi时SOD活性达最高值，表明侵染初期烟草疫霉已开始从伤口侵入钦果9号叶片。转录组分析共筛选获得7163个DEGs，KEGG信号通路富集分析发现，DEGs主要在植物激素信号转导、植物—病原菌互作、苯丙烷类生物合成、MAPK信号途径及类黄酮生物合成等与植物抗病相关的通路显著富集，表明这些通路在转录水平上是百香果响应烟草疫霉侵染的主要代谢途径。植物—病原菌互作通路中的PR1、CERK1、RPM1、CDPK和CML等基因，茉莉酸（JA）/乙烯（ET）信号途径中的JAR1、JAZ2、ERF1、EBF1和ERS等基因，以及次生代谢相关的CAD6、4CL、POD、CHS和LDOX等基因均在感染烟草疫霉的钦果9号植株中大幅上调表达，推测这些基因参与了百香果对烟草疫霉的抗性反应过程。实时荧光定量PCR结果验证了RNA-Seq数据的可靠性。【结论】植物激素信号转导、植物—病原菌互作、苯丙烷类生物合成、MAPK信号途径及类黄酮生物合成是百香果响应烟草疫霉侵染的主要信号通路和代谢途径。不同代谢途径中的关键基因以复杂方式参与了百香果对烟草疫霉的抗性反应过程。Abstract: 【Objective】The objective of this study was to analyze the expression patterns of passion fruit in response to the infection of Phytophthora nicotianae at the transcriptional level and identify the resistance-related genes and their mechanism， which provided a foundation for the screening of disease-resistance traits and breeding of disease-resistance varieties of passion fruit by molecular means. 【Method】The main cultivar of passion fruit Qinguo No. 9 was as the test material， and the tissue phenotype observation and physiological index determination of Qinguo No. 9 infected by P. nicotianae at 12， 24， 48， 72 and 96 hpi were carried out. The transcriptome of Qinguo No. 9 in response to P. nicotianae infection was constructed by transcriptome sequencing RNA-Seq. DESeq software was used to analyze the differences in gene expression， and differentially expressed genes（DEGs） were screened with the criteria of significant difference P<0.005， false discovery rate （FDR）≤0.001 and |log2 Ratio|≥1. GO functional annotation analysis and KEGG signaling pathway enrichment analysis of DEGs were carried out by BLAST2GO and KOBAS2 to clarify the main pathways and metabolism pathways of Qinguo No. 9 in response to P. nicotianae infection. The DEGs of metabolism pathways related to plant disease resistance were screened out and the gene expression patterns were analyzed with MeV software to construct the heat maps， and the dynamic change trend of DEGs in the process of P. nicotianae infecting Qinguo 9 was analyzed. The difference in gene expression was verified by real-time fluorescence quantitative PCR to be consistent with the transcriptome data. 【Result】There were no obvious symptoms in the early stage of infection in the leaves of Qinguo No. 9 by P. nicotianae， but the leaves appeared chlorotic at 48 hpi. The lesions became darker and expanded to obvious brown wilt spots accompany with infection time. The superoxide dismutase （SOD） activity in all treatments groups of Qinguo No. 9 were significantly higher than that of in control groups from 12 hpi（P<0.05）， and SOD activity reached the peak at 24 hpi. This indicated that P. nicotianae had begun to invade the leaves of Qinguo No. 9 from the wound at the early stage of infection. A total of 7163 DEGs were screened by transcriptome analysis. KEGG signaling pathway enrichment analysis showed that DEGs were mainly enriched in pathways of plant hormone signaling transduction， plant-pathogen interaction， phenylpropanoid biosynthesis， MAPK signaling pathway and flavonoid biosynthesis， which were related to plant disease resistance， indicating that these pathways may be the main metabolism pathways of passion fruit in response to P. nicotianae infection at transcriptional level. Genes of PR1， CERK1， RPM1， CDPK， CML in the plant-pathogen interaction pathway， genes of JAR1， JAZ2， ERF1， EBF1 and ERS in the jasmonic acid （JA）/ethylene （ET） signaling pathway， and secondary metabolism-related genes of CAD6， 4CL， POD， CHS， and LDOX were significantly up-regulated in Qinguo No. 9 infected with P. nicotianae. This suggested that these genes might be involved in the resistance of passion fruit to P. nicotianae. Real-time fluorescence quantitative PCR results verified the reliability of RNA-Seq data. 【Conclusion 】Plant hormone signaling transduction， plant-pathogen interaction， phenylpropanoid biosynthesis， MAPK signaling pathway and flavonoid biosynthesis are the main signaling and metabolism pathways of passion fruit against P. nicotianae infection. The key genes in different metabolism pathways are involved in resistance of passion fruit to P. nicotianae in complex ways.
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Keywords:
- passion fruit /
- Phytophthora nicotianae /
- transcriptome analysis /
- molecular mechanism /
- gene mining
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