刘彬,刘青华,周志春,徐六一,陈雪莲,罗柠.基于高通量转录组测序筛选马尾松抗松材线虫病相关基因[J].林业科学研究,2019,32(5):1-10
基于高通量转录组测序筛选马尾松抗松材线虫病相关基因
Identification of Candidate Constitutive Expressed Resistant Genes of Pine Wilt Disease in Pinus massoniana Based on High-throughput Transcriptome Sequencing
投稿时间:2018-11-26  修订日期:2019-03-11
DOI:10.13275/j.cnki.lykxyj.2019.05.001
中文关键词:  马尾松  松材线虫病  抗性  转录组测序  差异基因
英文关键词:Pinus massoniana  pine wilt disease  resistance  transcriptome sequencing  constitutive expressed genes
基金项目:“十三五”浙江省林木新品种选育重大专项课题(2016C02056-4);国家自然科学基金项目(31470670);江西省林业厅林业科技创新专项项目(201703);中央级公益性科研院所基本科研业务费专项资金项目(CAFYBB2017ZA001-2)
作者单位E-mail
刘彬 中国林业科学研究院亚热带林业研究所, 浙江省林木育种技术研究重点实验室, 浙江 杭州 311400  
刘青华 中国林业科学研究院亚热带林业研究所, 浙江省林木育种技术研究重点实验室, 浙江 杭州 311400 876009290@qq.com 
周志春 中国林业科学研究院亚热带林业研究所, 浙江省林木育种技术研究重点实验室, 浙江 杭州 311400  
徐六一 安徽省林业科学研究院, 安徽 合肥 230031  
陈雪莲 安徽省林业科学研究院, 安徽 合肥 230031  
罗柠 浙江省临海市自然资源和规划局, 浙江 临海 317000  
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中文摘要:
      [目的]研究马尾松抗松材线虫病相关基因,为解析马尾松抗性机理和高抗马尾松早期选择提供理论依据。[方法]对高抗和易感2种马尾松基因型接种松材线虫,在接种后第1、15和30天取样进行转录组高通量测序,通过对高抗和易感马尾松差异基因识别以及富集分析,以筛选与抗松材线虫病相关的组成型差异基因。[结果]对高抗和易感马尾松接种松材线虫后的转录组进行比较,在接种后第1、15和30天分别获得2 866、679和1 657个差异基因,且在接种松材线虫后不同时间点间,共有差异基因相对较少。对差异基因进行GO富集分析,结果显示:在接种后第1、15和30天,最显著的生物学进程是氧化-还原过程,而GO项刺激反应、转录调节以及香叶基二磷酸代谢过程也显著富集。对这几类GO项相关基因进一步分析,发现接种后3个时间点GO项刺激反应中共包括26个R基因,除了2个R基因外,其它R基因表达皆为组成型,在接种松材线虫和对照间差异不显著,而GO项转录调节中仅2个差异基因被nr注释为ERF转录因子,其余为未知基因。接种松材线虫后,ERF转录因子在高抗马尾松上的表达量始终高于易感马尾松上的表达量,且在接种松材线虫和对照间表达量变化不显著,也为组成型基因表达。GO项香叶基二磷酸代谢过程中,GGPPS 3个基因也在高抗马尾松上具有更高表达量,为组成型基因表达。这些基因中TIR-NBS-LRR基因(c65785.graph_c0)、ERF转录因子(c78073.graph_c0)和3个GGPPS基因在高抗马尾松中表达量较高,而在易感马尾松中表达量较低,甚至为0,可作为开发分子标记的候选基因用于鉴定抗性马尾松。[结论]高抗和易感马尾松在接种松材线虫后第1天其表达量差异达到显著水平的基因最多,其中,LRR基因、ERF转录因子和GGPPS与马尾松的抗性相关,部分TIR-NBS-LRR基因、ERF转录因子和GGPPS有望被开发为鉴定高抗马尾松的分子标记。
英文摘要:
      [Objective] To study the genes related to the resistance of Masson pine (Pinus massoniana) to pine wood nematode (Bursaphelenchus xylophilus, PWN).[Method] Based on two genotypes of resistant and susceptible P. massoniana for inoculating pine wood nematode, and sampling at the 1 dpi, 15 dpi and 30 dpi after inoculation, the authors performed a transcriptome analysis to identify differentially expressed constitutive genes associated with resistance to PWN infected.[Result] Comparing resistant and susceptible transcriptomes of P. massoniana inoculated with B. xylophilus, 2 866, 679 and 1 657 differentially expressed genes were obtained at 1, 15, or 30 days post-inoculation (dpi), and there were relatively few common differentially expressed genes at different time points. The GO enrichment analysis was performed on the differentially expressed genes, which indicated that the most significant biological process at the 1 dpi, 15 dpi and 30 dpi was oxidation-reduction process, while the stimulus response, transcriptional regulation and the geranyl diphosphate metabolic process in GO items were also significantly enriched. A further analysis of these related genes in the stimulus response GO term, 26 R genes were found at three time points after inoculation. The expression of the other R genes was constitutive except for two R genes, and no significant difference was observed between PWN and water inoculation. In the transcriptional regulation GO term, only two differentially expressed genes of ERF transcription factors were annotated by nr database, and the rest differentially expressed genes were unknown. After inoculation with PWN, the expression level of ERF transcription factors in resistant P. massoniana was always higher than that in susceptible ones, and there was insignificantly difference between trees inoculating PWN and the control. The result means that expression of the two ERF transcription factors is constitutive. In geranyl diphosphate metabolic process GO term, three GGPPS genes having higher expression level in high resistant P. massoniana, are constitutive expression genes. Here, the authors also found that the TIR-NBS-LRR gene (c65785.graph_c0), ERF transcription factor (c78073.graph_c0), and the three GGPPS genes, having higher expression in resistant P. massoniana, the expression levels were extremely low or even zero in susceptible P. massoniana, which suggested that these genes can be chosen as candidate genes for the development of molecular markers to identify resistant P. massoniana.[Conclusion] Most genes changed the expression level at 1 dpi between resistant and susceptible P. massoniana. The R genes, ERF transcription factors and GGPPS are related to the resistance of P. massoniana, some of which are expected to be developed as molecular markers for identifying PWN-resistance P. massoniana.
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