不同温度下TMV侵染枯斑三生烟的LncRNA差异表达

【目的】 筛选不同温度下烟草花叶病毒（Tobacco mosaic virus,TMV）侵染后枯斑三生烟（Nicotiana tabacum var. Samsun NN）差异表达的长链非编码RNA（long non-coding RNA,lncRNA）,研究lncRNA在枯斑三生烟抗性反应中的作用。【方法】 N基因的温度敏感性使枯斑三生烟在25℃时具备对TMV的抗性、在31℃抗性丧失,在这两个温度条件下对枯斑三生烟接种TMV和磷酸盐缓冲盐水（phosphate buffered saline,PBS）,48 h后提取系统叶总RNA,构建链特异性文库后进行深度测序。对测序结果进行过滤后利用HTSeq将有效数据与近缘品种TN90（N. tabacum var. TN90）基因组比对,筛选得到lncRNA后利用FPKM法估计lncRNA的表达水平。通过edgeR筛选差异表达lncRNA（differentially expressed lncRNA,DElncRNA）,并利用qRT-PCR技术对这一结果进行验证。通过共定位及共表达分析预测DElncRNA的靶基因,通过参考基因组注释、GO和KEGG富集分析研究靶基因的功能。【结果】 4个处理共12个样本经lncRNA-seq各测得约8 000万条clean reads,共获得4 737条已知lncRNA、40 169条新lncRNA。其中64个lncRNA在不同温度条件下TMV侵染后存在差异表达,qRT-PCR测定结果显示这些lncRNA的测序正确率在80%左右,表明本研究所得测序数据具备较高的可信度。对DElncRNA进行靶基因预测,发现一些基因同时被25℃下调和31℃上调的DElncRNA靶向。靶基因注释功能丰富,主要参与植物抗病、激素和代谢等生理过程。部分可能与激素通路相关的lncRNA,在25℃下TMV侵染时呈现下调趋势,而在31℃下TMV侵染则呈现上调趋势。GO富集分析显示靶基因主要参与构成膜、囊泡等组分,具备钙、钾离子通道抑制剂活性等分子功能,使相应离子得以转运引发随后的反应,同时也参与发病、抗原加工和呈现、细胞分裂素代谢等生理过程。KEGG分析发现靶基因显著富集在植物激素信号转导通路,25℃下调和31℃上调的DElncRNA靶基因同时富集在激素信号传导、ABC运输蛋白、苯丙烷类生物合成等通路。【结论】 不同温度（25℃和31℃）条件下TMV侵染枯斑三生烟后,长链非编码RNA差异表达,DElncRNA通过作用于激素信号传导、物质转运等过程参与寄主系统获得性抗性反应。研究结果可为揭示植物系统获得性抗性中lncRNA的调控功能以及新型抗病毒技术开发提供依据。

Differential Expression of LncRNAs in Nicotiana tabacum var. Samsun NN Infected by TMV at Different Temperatures

【Objective】 The objective of this study is to screen out the differentially expressed lncRNAs in Nicotiana tabacum var. Samsun NN after Tobacco mosaic virus (TMV) infection at different temperatures, and to investigate the role of identified lncRNAs in Samsun NN’s resistance response.【Method】 The temperature sensitivity of N gene makes Samsun NN have the resistance to TMV at 25℃, but lose it at 31℃. TMV and phosphate buffered saline (PBS) were mechanically inoculated into Samsun NN at 25℃ and 31℃. Total RNAs were extracted from systemic leaves at 48 hpi (hours post infection). Deep sequencing was performed after strand-specific database construction, and the sequencing results were filtered to get clean reads. Using N. tabacum var. TN90 as a reference, HTseq was employed to compare obtained reads. LncRNAs were screened out, and their expression levels were estimated by FPKM method. Differentially expressed lncRNAs (DElncRNAs) were then identified by edgeR and verified by qRT-PCR. The target genes of DElncRNAs were predicted by co-localization and co-expression analyses. Gene annotations, GO and KEGG pathways were analyzed for functional prediction of target genes. 【Result】 Altogether, 80 million clean reads were detected for each of 12 samples from 4 treatments by lncRNA-seq. A total of 4 737 annotated lncRNAs and 40 169 novel lncRNAs were obtained. Among them, 64 lncRNAs were differentially expressed after TMV infection at different temperatures. qRT-PCR results showed that the sequencing accuracy of these DElncRNAs was about 80%, which indicated the sequencing data obtained in this study had high reliability. Importantly, some target genes were simultaneously targeted by DElncRNAs that were down-regulated at 25℃ and up-regulated at 31℃. Gene annotations showed that DElncRNAs’ target genes involved in many functions, such as plant resistance, hormone and metabolic pathways. Particularly, some lncRNAs that may be associated with hormone pathways showed a down-regulation trend after TMV infection at 25℃, while up-regulated at 31℃. Furthermore, GO enrichment analysis showed that target genes were mainly involved in the composition of membranes, vesicles, and acted as calcium and potassium ion channel inhibitor activity, so that consequently the corresponding ions could be transported to their sites of action to trigger subsequent reactions. Moreover, these genes were also involved in pathogenesis, antigen processing and presentation, cytokinin metabolism and other physiological processes. The plant hormone signaling pathway was significantly enriched by target genes during KEGG pathway analysis. DElncRNAs related genes down-regulated at 25℃ and up-regulated at 31℃ were simultaneously enriched in pathways such as hormone signaling, ABC transporters, and phenylpropanoid biosynthesis.【Conclusion】 LncRNAs were differentially expressed in Samsun NN after TMV infection at different temperatures (25℃ and 31℃). DElncRNAs participated in host plant systemic acquired resistance by acting on hormone signaling transduction, substance transport and other processes. Taken together, this study lays a foundation for further research on lncRNA’s regulatory function in plant systemic acquired resistance and the development of new strategies to overcome virus invasion into host plant.