甘薯病毒病原相关的microRNA的挖掘及分析

doi:10.3864/j.issn.0578-1752.2018.11.007

摘要/Abstract

摘要： 【目的】甘薯的病毒种类比较多，不同的病毒感染会引起相应的症状变化，田间自然发病的甘薯表型不一，这可能与病毒感染类型有关。论文旨在鉴定和研究甘薯中与不同甘薯病毒感染相关的microRNA（miRNA）。【方法】采用Illumina公司高通量测序技术，对来自福建泉州地区同一品种（龙薯9号）、具有不同症状的叶片样本（畸形黄化、疱疹、褪绿矮化和曲叶，样本编号分别为Fj01、Fj02、Fj03和1H）进行高通量测序，利用生物信息学手段挖掘和分析差异表达miRNA，并对差异miRNA和病毒表达进行PCR验证，鉴定基因和病毒在样本中表达情况，分析测序数据的可靠性，利用生物信息学分析进行miRNA靶基因预测和功能分析，探究差异miRNA与病毒种类的相关性。【结果】通过与病毒数据库的比对，发现Fj01、Fj02、Fj03样本（除1H外）均感染了甘薯常见病毒，如甘薯褪绿矮化病毒（Sweet potato chlorotic stunt virus，SPCSV）、甘薯病毒2号（Sweet potato virus 2，SPV2）、甘薯羽状斑驳病毒（Sweet potato feathery mottle virus，SPFMV）、甘薯G病毒（Sweet potato virus G，SPVG）、甘薯C病毒（Sweet potato virus C，SPVC）等，但Fj01、Fj02和Fj03这3个样本的症状并不一致，样本之间只在甘薯不常见的病毒种类中有差异，通过PCR验证了SPFMV和SPVC病毒在样本中的表达情况与测序数据基本一致。在4个样本中共鉴定出679个已知的miRNA和1 004个新的miRNA，通过配对比较分析，其中288个已知的miRNA和433个新的miRNA在4个样本之间有差异性表达，并且这些miRNAs，如miR-156、miR-157、miR-166等家族的成员在4个样本中表达模式各异。同时，采用实时荧光定量PCR验证几个差异miRNAs（如miR-156、novel-miR-40和miR-319m）在各个样本间的表达情况与测序结果基本一致，另外，与脱毒苗样本进行比较，检测到3个miRNA（miR-160a、miR-2096和miR-5387b）在4个症状样本中具有不同程度的上调表达，证明miRNA的表达与植株的表型有关。通过对miRNA靶基因的预测与分析，发现这些差异miRNA的靶基因多数为转录调控因子，编码蛋白多含有ZFP、WD、Myb、SPL等功能区域，这些因子多参与调控植物的基因、代谢通路和抗原识别等来调控植物生长发育、形态建成和抗逆性，表明这些miRNA靶基因的功能多样性。【结论】不同病毒感染确实能够引起miRNA差异表达，并且这些miRNA通过其靶基因参与调控植物的生长发育、抗胁迫性和防御。

关键词: 甘薯, microRNA, 高通量测序, 甘薯病毒

Abstract: 【Objective】Various viruses had been identified from sweet potato. Infections with different sweet potato viruses may result in different pathological symptoms of plants. The objective of this study is to investigate the microRNAs (miRNAs) in response to different pathogens from sweet potato viruses in sweet potato.【Method】Illumina RNA sequencing was performed to mine and identify the differentially expressed miRNAs among samples with different infection pathological symptoms (yellowing and deformity, herpes, chlorotic and dwarf, and curl; sample was named Fj01, Fj02, Fj03 and 1H, respectively) of ‘Longshu 9’ from Quanzhou, Fujian, China. Total and differentially expressed miRNAs among samples were identified. Real-time PCR analysis of several miRNAs and viruses was performed to validate the quality of sequencing data. Target predication and functional enrichment for these miRNAs were analyzed. Association between miRNAs and virus infections would be discussed.【Result】 After the alignment to the virus database of sweet potato, 3 samples (without 1H) were simultaneously infected with common sweet potato viruses, including Sweet potato chlorotic stunt virus (SPCSV), Sweet potato virus 2 (SPV2), Sweet potato feathery mottle virus (SPFMV), Sweet potato virus G (SPVG), and Sweet potato virus C (SPVC), although they showed different pathological symptoms. In addition, uncommon viruses were identified in different samples. Using PCR analysis of SPFMV and SPVC viruses, the consistence with sequencing data were validated. Using sequencing analysis, 679 known miRNAs and 1 004 novel miRNAs were found in these 4 samples. With comparative analysis, it was identified that 288 known miRNAs and 433 novel miRNAs were differentially expressed among these 4 samples. These miRNAs showed different expression profiles among samples, such as the members of miR-156, miR-157, and miR-166 families. qRT-PCR analysis showed these expression profiles of several miRNAs (including miR-156, novel-miR-40 and miR-319m) were in consistence with their profiles by sequencing. In comparison with that in virus-free seedling, the expression of 3 miRNAs (miR-160a, miR-2096 and miR-5387b) was differentially upregulated in these 4 sequencing samples. These results suggested that these miRNAs might be related to virus infections. Further target predication found that most of these miRNA targets were transcription factors, including genes encoding ZFP, WD, Myb, and SPL domain-containing proteins. Enrichment analysis for those targets showed that they were associated with plant growth and development, morphogenesis, and stress resistance via regulating genes, signaling pathways, and antigen recognition. These represented the multipotent functions of these miRNAs’ targets.【Conclusion】Different sweet potato viruses induced differences in miRNA profiling, and these miRNAs were mainly associated with plant growth, resistance and plant defense.

Key words: sweet potato, microRNA, Illumina sequencing, sweet potato virus

李华伟，刘中华，张鸿，许泳清，李国良，林赵淼，邱永祥，罗文彬，纪荣昌，汤浩，邱思鑫. 甘薯病毒病原相关的microRNA的挖掘及分析[J]. 中国农业科学, 2018, 51(11): 2094-2105.

LI HuaWei, LIU ZhongHua, ZHANG Hong, XU YongQing, LI GuoLiang, LIN ZhaoMiao, QIU YongXiang, LUO WenBin, JI RongChang, TANG Hao, QIU SiXin. Mining and Characterization of MicroRNAs Associated with Pathogenicity by Different Sweet Potato Viruses[J]. Scientia Agricultura Sinica, 2018, 51(11): 2094-2105.

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