中国农业科学 ›› 2019, Vol. 52 ›› Issue (21): 3794-3805.doi: 10.3864/j.issn.0578-1752.2019.21.008

• 植物保护 • 上一篇    下一篇

本生烟响应蛋白激发子PevD1的差异表达基因鉴定与分析

梁颖博,李泽,邱德文,曾洪梅,李广悦,杨秀芬()   

  1. 中国农业科学院植物保护研究所植物病虫害生物学国家重点实验室,北京100081
  • 收稿日期:2019-05-22 接受日期:2019-06-28 出版日期:2019-11-01 发布日期:2019-11-12
  • 通讯作者: 杨秀芬
  • 作者简介:梁颖博,E-mail: Lyingbo16@163.com
  • 基金资助:
    国家重点研发计划(2017YFD0201101);国家自然科学基金(31772151)

Identification and Analysis of Differentially Expressed Genes Induced by Protein Elicitor PevD1 in Nicotiana benthamiana

LIANG YingBo,LI Ze,QIU DeWen,ZENG HongMei,LI GuangYue,YANG XiuFen()   

  1. State Key Laboratory for Biology of Plant Disease and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100081
  • Received:2019-05-22 Accepted:2019-06-28 Online:2019-11-01 Published:2019-11-12
  • Contact: XiuFen YANG

摘要:

目的 通过RNA-Seq筛选本生烟(Nicotiana benthamiana)响应大丽轮枝菌(Verticillium dahliae)蛋白激发子PevD1的差异表达基因(differentially expressed gene,DEG),分析PevD1诱导植物产生抗病性的潜在分子机制。方法 用10 μmol·L -1的PevD1蛋白液渗入4周龄的本生烟叶片,分别在处理后6、12和24 h取样提取RNA,构建mRNA文库后采用BGISEQ-500平台进行测序。筛选各时间点的差异表达基因进行GO和KEGG分析;重点分析与诱导抗病相关的富含亮氨酸重复序列类受体蛋白激酶(leucine-rich repeats RLKs,LRR-RLKs)、转录因子(transcription factor,TF)以及病程相关蛋白(pathogenesis related protein,PR蛋白)家族差异表达基因;采用qRT-PCR对差异表达基因进行定量验证。 结果 GO功能富集以及KEGG通路富集分析表明,PevD1诱导6 h后的差异表达基因主要与细胞识别、光合作用、光收割等功能相关,显著富集在光合作用-天线蛋白通路、萜类化合物合成通路、黄酮和黄酮醇等次生代谢产物合成相关通路中;12 h和24 h的差异表达基因主要与细胞识别和胞内激酶等生物学功能相关,显著富集在植物-病原互作通路、倍半萜和三萜生物合成通路、黄酮和黄酮醇生物合成通路、亚麻酸代谢等次生代谢产物合成相关通路中。与光合作用相关的差异表达基因主要呈下调趋势,与萜类、黄酮类等抗病相关次生代谢产物合成通路相关的差异表达基因主要呈上调趋势。PevD1诱导后大量的LRR-RLKs、TF以及PR蛋白家族基因显著上调表达,这些基因与激发子识别、基因转录调控和抗病性相关。经qRT-PCR验证后,所检测基因的表达趋势与转录组测序结果一致。结论 PevD1诱导本生烟中大量基因转录重排,大量LRR-RLKs、TF和PR蛋白家族基因上调表达,激活了植物免疫系统,使植物产生抗病性。研究结果可为今后深入探讨PevD1诱导植物免疫的机理提供依据。

关键词: 本生烟, 大丽轮枝菌, 蛋白激发子PevD1, RNA-Seq, LRR-RLKs, 转录因子, PR蛋白

Abstract:

【Objective】 The objective of this study is to screen the differentially expressed genes (DEGs) induced by protein elicitor PevD1 of Verticillium dahliae in Nicotiana benthamiana by RNA-Seq, and to analyze the potential mechanism of PevD1-induced disease resistance. 【Method】 Leaves of 4-week-old N. benthamiana were infiltrated with 10 μmol·L -1 PevD1 solution, and samples were taken at 6, 12 and 24 h after PevD1 treatment, then RNA was extracted. The mRNA libraries were constructed and sequenced by BGISEQ-500 platform. The DEGs at each time point were screened for GO and KEGG analysis. The leucine-rich repeat receptor-like kinases (LRR-RLKs), transcription factors (TFs) and pathogenesis-related (PR) proteins family in DEGs were analyzed, and qRT-PCR was used to verify the expression levels of relevant DEGs.【Result】 GO functional enrichment and KEGG pathway enrichment analysis indicated that the DEGs at 6 h post infiltration (hpi) were mainly related to cell recognition, photosynthesis, light-harvesting, and were significantly enriched in photosynthesis-antenna protein pathways, terpenoid synthesis pathway, flavonoid and flavonol, and other secondary metabolite synthesis pathways. The DEGs at 12 hpi and 24 hpi were mainly associated with cell recognition and biological functions such as intracellular kinase activity, and were significantly enriched in plant-pathogen interaction pathway, sesquiterpene and triterpenoid biosynthetic pathway, flavonoid and flavonol biosynthetic pathway, linolenic acid metabolism. The unigenes in photosynthesis-antenna proteins, photosynthesis and porphyrin, chlorophyll metabolism pathway were mainly down-regulated, and the unigenes in sesquiterpenoid and triterpenoid biosynthesis, flavone and flavonol biosynthesis, plant-pathogen interaction, phenylpropanoid biosynthesis and terpenoid backbone biosynthesis pathway were mainly up-regulated. After PevD1 induction, a large number of LRR-RLKs, TFs and PR proteins family genes were significantly up-regulated, which were related to elicitor recognition, gene transcriptional regulation and disease resistance. qRT-PCR analysis showed that the expression pattern of the detected DEGs was consistent with the RNA-Seq results.【Conclusion】 PevD1 induced a large number of gene transcriptional rearrangements in N. benthamiana. Lots of LRR-RLKs, TFs and PR proteins family genes were up-regulated, which activated the plant immune system and conferred to plants disease resistant. These results can provide a basis for further study on the mechanism of PevD1-induced immunity in the future.

Key words: Nicotiana benthamiana, Verticillium dahliae, protein elicitor PevD1, RNA-Seq, LRR-RLKs, transcription factor, PR protein